Structured Review

Roche proteinase k
Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and <t>Proteinase</t> K prior to gel electrophoresis. Scale bar in B and C is 25 μm.
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Images

1) Product Images from "TF-ChIP Method for Tissue-Specific Gene Targets"

Article Title: TF-ChIP Method for Tissue-Specific Gene Targets

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2019.00095

Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.
Figure Legend Snippet: Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

Techniques Used: Expressing, Immunofluorescence, Transgenic Assay, Activation Assay, Staining, Sonication, Nucleic Acid Electrophoresis

2) Product Images from "The Biomechanical Properties of Mixtures of Blood and Synovial Fluid"

Article Title: The Biomechanical Properties of Mixtures of Blood and Synovial Fluid

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

doi:

An HA ladder (lane 1), bSF (lane 2), and portions of blood/SF clots digested with proteinase K (lanes 3–7) were electrophoresed on a 1% agarose gel and stained with Stains-all.
Figure Legend Snippet: An HA ladder (lane 1), bSF (lane 2), and portions of blood/SF clots digested with proteinase K (lanes 3–7) were electrophoresed on a 1% agarose gel and stained with Stains-all.

Techniques Used: Agarose Gel Electrophoresis, Staining

3) Product Images from "Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella"

Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-13-80

Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the  nirB  promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the  nirB  and  spiC  promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of  Salmonella  strain SL7207 pZ1204.
Figure Legend Snippet: Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the nirB promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the nirB and spiC promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of Salmonella strain SL7207 pZ1204.

Techniques Used: Expressing, Western Blot

Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.
Figure Legend Snippet: Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.

Techniques Used: Western Blot, Expressing

4) Product Images from "The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x"

Article Title: The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200701005

Endogenous Sam68 associates with the mRNAs encoding for regulators of apoptosis. 1 mg of HEK293 cell extracts was immunoprecipitated with 10 μg of either control rabbit IgGs or anti-Sam68 IgGs as described in Materials and methods. An aliquot of the immunoprecipitated proteins was analyzed in Western blot for the presence of Sam68 (A), and the remaining sample was extracted in phenol/chloroform after treatment with proteinase K and DNase. (B) Extracted RNA was retrotranscribed and used for PCR amplification with oligonucleotides specific for the indicated genes. (C) GST pull-down experiment using purified GST or GST-Sam68 immobilized on glutathione-agarose beads and 100 μg of total RNA extracted and purified from HEK293 cells. Adsorbed RNA was extracted, retrotranscribed, and amplified as described in B.
Figure Legend Snippet: Endogenous Sam68 associates with the mRNAs encoding for regulators of apoptosis. 1 mg of HEK293 cell extracts was immunoprecipitated with 10 μg of either control rabbit IgGs or anti-Sam68 IgGs as described in Materials and methods. An aliquot of the immunoprecipitated proteins was analyzed in Western blot for the presence of Sam68 (A), and the remaining sample was extracted in phenol/chloroform after treatment with proteinase K and DNase. (B) Extracted RNA was retrotranscribed and used for PCR amplification with oligonucleotides specific for the indicated genes. (C) GST pull-down experiment using purified GST or GST-Sam68 immobilized on glutathione-agarose beads and 100 μg of total RNA extracted and purified from HEK293 cells. Adsorbed RNA was extracted, retrotranscribed, and amplified as described in B.

Techniques Used: Immunoprecipitation, Western Blot, Polymerase Chain Reaction, Amplification, Purification

5) Product Images from "Alu RNP and Alu RNA regulate translation initiation in vitro"

Article Title: Alu RNP and Alu RNA regulate translation initiation in vitro

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl246

Alu RNP purification on Superdex 200 . ( A ) Denaturing acrylamide gel. Synthetic Alu RNA migrates as a single band with the expected size of 305 nt. ( B ) Native acrylamide gel. Alu RNA migrates in a defined band indicating that it is homogeneously folded. Trace amounts of an RNA dimer are also observed (star). ( C ) OD 254 elution profile of a Superdex 200 column. Free Alu RNA, blue; Alu RNA bound to recombinant SRP9/14, red. Fractions 7–10 (grey box) containing Alu RNA in complex with two SRP9/14 proteins were pooled for subsequent experiments. The second RNP peak most likely represents Alu RNA bound to one protein and free RNA. ( D ) Aliquots of 1 and 2 µl (I and II, respectively) of the purified RNP fraction were subjected to denaturing acrylamide gel electrophoresis after proteinase K digestion. ( E ) Aliquots of 1 and 2 µl (I and II, respectively) of the purified RNP fraction were subjected to immunoblotting with anti-SRP14 antibodies. ( F ) Native agarose gel electrophoresis of the purified RNP fraction and free RNA.
Figure Legend Snippet: Alu RNP purification on Superdex 200 . ( A ) Denaturing acrylamide gel. Synthetic Alu RNA migrates as a single band with the expected size of 305 nt. ( B ) Native acrylamide gel. Alu RNA migrates in a defined band indicating that it is homogeneously folded. Trace amounts of an RNA dimer are also observed (star). ( C ) OD 254 elution profile of a Superdex 200 column. Free Alu RNA, blue; Alu RNA bound to recombinant SRP9/14, red. Fractions 7–10 (grey box) containing Alu RNA in complex with two SRP9/14 proteins were pooled for subsequent experiments. The second RNP peak most likely represents Alu RNA bound to one protein and free RNA. ( D ) Aliquots of 1 and 2 µl (I and II, respectively) of the purified RNP fraction were subjected to denaturing acrylamide gel electrophoresis after proteinase K digestion. ( E ) Aliquots of 1 and 2 µl (I and II, respectively) of the purified RNP fraction were subjected to immunoblotting with anti-SRP14 antibodies. ( F ) Native agarose gel electrophoresis of the purified RNP fraction and free RNA.

Techniques Used: Purification, Acrylamide Gel Assay, Recombinant, Electrophoresis, Agarose Gel Electrophoresis

6) Product Images from "Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing"

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing

Journal: Scientific Reports

doi: 10.1038/srep24228

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the  32 P-labeled 5′-end of the 5 S  ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S  DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S  ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.
Figure Legend Snippet: Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the 32 P-labeled 5′-end of the 5 S ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.

Techniques Used: Labeling, Tube Formation Assay, Incubation, Agarose Gel Electrophoresis

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.

Techniques Used: Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

7) Product Images from "Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing"

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing

Journal: Scientific Reports

doi: 10.1038/srep24228

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with a mono-nucleosome. ( a ) Scheme of the ternary complex formation assay. ( b, c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with ssDNA-conjugated magnetic beads (final 5 μM in nucleotides). A heterologous poly dT 80-mer or a homologous ssDNA 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–8) or mono-nucleosomes (lanes 9–16) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, 9, and 13 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the  32 P-labeled 5′-end of the 5 S  ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S  DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S  ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.
Figure Legend Snippet: Competitive homologous-pairing assay. ( a ) Scheme of the competitive homologous-pairing assay. Asterisks indicate the 32 P-labeled 5′-end of the 5 S ssDNA 70-mer. ( b ) The D-loop formation assay on the 5 S DNA sequences. RAD51 (0.4 μM) or DMC1 (0.4 μM) was incubated with the 5 S ssDNA 70-mer (final 1 μM in nucleotides). The reactions were conducted in the presence of HOP2-MND1 (denoted as H2M1). The reaction was initiated by adding the naked dsDNA (final 30 μM in nucleotides) in the presence of the competitor nucleosome. The nucleosome concentrations are indicated at the top of the panel. After a 10 min incubation, the reaction was stopped by SDS and proteinase K, and the reaction products were separated by agarose gel electrophoresis. ( c ) Graphic representation of the experiments shown in panel ( b ). The amounts of D-loop formation relative to RAD51 or DMC1 with HOP2-MND1 (lanes 2 and 7 of panel ( b )) are plotted against the nucleosome concentrations. The average values of four independent experiments are shown with the SD values.

Techniques Used: Labeling, Tube Formation Assay, Incubation, Agarose Gel Electrophoresis

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with the nucleosome lacking the N-terminal histone tails. ( a ) RAD51 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). A heterologous poly dT 80-mer was used as the ssDNA substrate. HOP2-MND1 (denoted as H2M1) was then added to the reaction mixtures. After a 5 min incubation, naked dsDNA (lanes 1–4), wild-type mono-nucleosomes (lanes 5–8), or all tailless mono-nucleosomes (lanes 9–12) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The asterisk indicates poly dT 80-mer ssDNA. The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 5, and 9 were performed in the absence of RAD51 and DMC1. ( b ) Graphic representation of the experiments shown in panel ( a ). The amounts of the ternary complex formation are plotted against the RAD51 concentration. The average values of three independent experiments are shown with the SD values. ( c ) DMC1 (1.7, 3.4, and 6.8 μM) was incubated with the ssDNA-conjugated magnetic beads (final 20 μM in nucleotides). The experiments were performed by the same procedure as in panel ( a ). ( d ) Graphic representation of the experiments shown in panel ( c ). The amounts of the ternary complex formation are plotted against the DMC1 concentration. The average values of three independent experiments are shown with the SD values.

Techniques Used: Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining, Concentration Assay

Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.
Figure Legend Snippet: Ternary complex formation by the RAD51-ssDNA and DMC1-ssDNA complexes with nucleosome arrays. ( a ) Scheme of the ternary complex formation assay with the nucleosome arrays. ( b,c ) RAD51 or DMC1 (0.7, 1.3, and 1.7 μM) was incubated with the ssDNA (poly dT 80-mer)-conjugated magnetic beads in the presence of HOP2-MND1 (denoted as H2M1). After a 5 min incubation, naked dsDNA (lanes 1–5), tri-nucleosomes (lanes 6–10), or di-nucleosomes (lanes 11–15) were added to each reaction mixture. The naked and nucleosomal dsDNA concentrations were 10 μM in nucleotides. The naked or nucleosomal dsDNA captured by the RAD51-ssDNA or DMC1-ssDNA complex was treated with SDS and proteinase K, and the samples were subjected to non-denaturing polyacrylamide gel electrophoresis (top panel). The naked and nucleosomal dsDNAs in the unbound fractions were also treated with SDS and proteinase K, and the samples (20%) were analyzed by non-denaturing polyacrylamide gel electrophoresis (middle panel). Bands were visualized by SYBR Gold staining. The reactions in lanes 1, 6, and 11 were performed in the absence of RAD51 and DMC1. The average values of three independent experiments are shown in the bottom panel, with the SD values. Panels ( b , c ) represent experiments with RAD51 and DMC1, respectively.

Techniques Used: Tube Formation Assay, Incubation, Magnetic Beads, Polyacrylamide Gel Electrophoresis, Staining

8) Product Images from "Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies"

Article Title: Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

Journal: Nature Communications

doi: 10.1038/ncomms11734

The 7D10 antibody triggers mitochondrial outer membrane permeabilization by binding to the α1–α2 loop of human Bak. ( a ) The 7D10 antibody induces cytochrome c release. Membrane fractions from Bak −/− Bax −/− MEFs, those cells expressing human Bak (hBak), or Bax −/− MEFs, were incubated with tBid or with the 7D10 or 8F8 antibodies. Supernatant (Sup) and pellet (Mito) fractions were assessed for cytochrome c release. ( b ) 7D10 triggers Bak conformational change as indicated by susceptibility to proteinase K. Incubations from a were treated with proteinase K and immunoblotted for Bak. Note that 7D10 binding at the loop masks a cleavage site (lane 4, Supplementary Fig. 1a ), and that uncleaved Bak and light chain co-migrate. ( c ) 7D10 triggers Bak oligomerization. Incubations from a were treated with oxidant (CuPhe) to induce disulfide bond formation and immunoblotted for Bak. M, monomer; M x, intramolecular linked monomers; D, intermolecular linked dimers. ( d ) The 7D10 trigger site in Bak is distinct from the canonical BH3-only trigger site. Cartoon representation of BakΔN19ΔC25 (2IMT, white) highlighting the α1–α2 loop (blue), and α3 and α4 of the hydrophobic groove (green). ( e ) Mutation of Bak G51 or P55 inhibits binding by 7D10. Membrane fractions from Bak −/− Bax −/− MEFs expressing the indicated hBak variants were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. IP, immunoprecipitated; UB, unbound; #, light chain. ( f ) Mutation of Bak G51 or P55 prevent Bak activation and cytochrome c release by 7D10. Membrane fractions from e were incubated with tBid or 7D10 and assessed for cytochrome c release. ( g ) Substitutions in mouse Bak to generate the 7D10 epitope. ( h ) The 51 GVAAPAD 57 sequence allows binding of 7D10 to mouse Bak. Membrane fractions from Bax −/− MEFs (mBak cells), or Bak −/− Bax −/− MEFs expressing hBak or the indicated mBak variants, were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. ( i ) The 51 GVAAPAD 57 sequence allows 7D10 to activate mouse Bak and release cytochrome c . Membrane fractions from h were incubated with tBid or 7D10 and assessed for cytochrome c release. In a – c , e , f , h and i data are representative of three independent experiments.
Figure Legend Snippet: The 7D10 antibody triggers mitochondrial outer membrane permeabilization by binding to the α1–α2 loop of human Bak. ( a ) The 7D10 antibody induces cytochrome c release. Membrane fractions from Bak −/− Bax −/− MEFs, those cells expressing human Bak (hBak), or Bax −/− MEFs, were incubated with tBid or with the 7D10 or 8F8 antibodies. Supernatant (Sup) and pellet (Mito) fractions were assessed for cytochrome c release. ( b ) 7D10 triggers Bak conformational change as indicated by susceptibility to proteinase K. Incubations from a were treated with proteinase K and immunoblotted for Bak. Note that 7D10 binding at the loop masks a cleavage site (lane 4, Supplementary Fig. 1a ), and that uncleaved Bak and light chain co-migrate. ( c ) 7D10 triggers Bak oligomerization. Incubations from a were treated with oxidant (CuPhe) to induce disulfide bond formation and immunoblotted for Bak. M, monomer; M x, intramolecular linked monomers; D, intermolecular linked dimers. ( d ) The 7D10 trigger site in Bak is distinct from the canonical BH3-only trigger site. Cartoon representation of BakΔN19ΔC25 (2IMT, white) highlighting the α1–α2 loop (blue), and α3 and α4 of the hydrophobic groove (green). ( e ) Mutation of Bak G51 or P55 inhibits binding by 7D10. Membrane fractions from Bak −/− Bax −/− MEFs expressing the indicated hBak variants were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. IP, immunoprecipitated; UB, unbound; #, light chain. ( f ) Mutation of Bak G51 or P55 prevent Bak activation and cytochrome c release by 7D10. Membrane fractions from e were incubated with tBid or 7D10 and assessed for cytochrome c release. ( g ) Substitutions in mouse Bak to generate the 7D10 epitope. ( h ) The 51 GVAAPAD 57 sequence allows binding of 7D10 to mouse Bak. Membrane fractions from Bax −/− MEFs (mBak cells), or Bak −/− Bax −/− MEFs expressing hBak or the indicated mBak variants, were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. ( i ) The 51 GVAAPAD 57 sequence allows 7D10 to activate mouse Bak and release cytochrome c . Membrane fractions from h were incubated with tBid or 7D10 and assessed for cytochrome c release. In a – c , e , f , h and i data are representative of three independent experiments.

Techniques Used: Binding Assay, Expressing, Incubation, Mutagenesis, Immunoprecipitation, Activation Assay, Sequencing

9) Product Images from "Interaction and uptake of exosomes by ovarian cancer cells"

Article Title: Interaction and uptake of exosomes by ovarian cancer cells

Journal: BMC Cancer

doi: 10.1186/1471-2407-11-108

Effect of proteinase K treatment in SKOV3 exosomes uptake . (A) Exos-CFSE (20 μg protein; green) or (B) SKOV3 cells were treated with 100 μg/ml proteinase K for 30 min. Uptake was determined after 4 h of incubation by flow cytometry analysis and compared with uptake of Exos-CFSE with no treatment (solid lines). Unlabelled SKOV3 cells (grey) were used as control for cell autofluorescence. Dashed lines represent Exos-CFSE uptake after proteinase K digestion. The results shown are representative of three independent experiments performed in duplicate.
Figure Legend Snippet: Effect of proteinase K treatment in SKOV3 exosomes uptake . (A) Exos-CFSE (20 μg protein; green) or (B) SKOV3 cells were treated with 100 μg/ml proteinase K for 30 min. Uptake was determined after 4 h of incubation by flow cytometry analysis and compared with uptake of Exos-CFSE with no treatment (solid lines). Unlabelled SKOV3 cells (grey) were used as control for cell autofluorescence. Dashed lines represent Exos-CFSE uptake after proteinase K digestion. The results shown are representative of three independent experiments performed in duplicate.

Techniques Used: Incubation, Flow Cytometry, Cytometry

10) Product Images from "Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿ †"

Article Title: Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿Proteomic Characterization of Pseudorabies Virus Extracellular Virions ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.02253-10

Peptide sequence coverage of viral proteins in untreated and proteinase K-treated virions. Schematic representations of each protein are oriented with the N terminus on the left and the C terminus on the right. Regions of the viral proteins that correspond
Figure Legend Snippet: Peptide sequence coverage of viral proteins in untreated and proteinase K-treated virions. Schematic representations of each protein are oriented with the N terminus on the left and the C terminus on the right. Regions of the viral proteins that correspond

Techniques Used: Sequencing

Identification of viral and host virion proteins. (A) Mock, proteinase K-treated, or untreated purified virions prepared from 75-cm 2  PK15 cells that were PRV Becker infected or mock infected. Samples were run on a 4 to 12% 1D SDS-PAGE gel and stained
Figure Legend Snippet: Identification of viral and host virion proteins. (A) Mock, proteinase K-treated, or untreated purified virions prepared from 75-cm 2 PK15 cells that were PRV Becker infected or mock infected. Samples were run on a 4 to 12% 1D SDS-PAGE gel and stained

Techniques Used: Purification, Infection, SDS Page, Staining

Sensitivity of viral glycoproteins gC, gB, gI, and gB to proteinase K. Western blot analysis of viral proteins in cell lysates from mock-infected and PRV Becker-infected PK15 cells, and purified virions from mock, untreated, and proteinase K (ProK)-treated
Figure Legend Snippet: Sensitivity of viral glycoproteins gC, gB, gI, and gB to proteinase K. Western blot analysis of viral proteins in cell lysates from mock-infected and PRV Becker-infected PK15 cells, and purified virions from mock, untreated, and proteinase K (ProK)-treated

Techniques Used: Western Blot, Infection, Purification

Functional classification of host proteins in PRV virions. We identified a total of 48 host proteins that were present in untreated and proteinase K-treated virions but not in samples from mock-infected cells. These proteins were manually classified according
Figure Legend Snippet: Functional classification of host proteins in PRV virions. We identified a total of 48 host proteins that were present in untreated and proteinase K-treated virions but not in samples from mock-infected cells. These proteins were manually classified according

Techniques Used: Functional Assay, Infection

11) Product Images from "Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells"

Article Title: Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

Journal: Eukaryotic Cell

doi: 10.1128/EC.4.11.1951-1958.2005

SDS-PAGE of extracted LPG from parent (PA) and mutant parasites. LPG was prepared by sonication of parasites, DNase and RNase treatment, followed by proteinase K digestion and phenol extraction. The aqueous phase was concentrated and extracted with solvent
Figure Legend Snippet: SDS-PAGE of extracted LPG from parent (PA) and mutant parasites. LPG was prepared by sonication of parasites, DNase and RNase treatment, followed by proteinase K digestion and phenol extraction. The aqueous phase was concentrated and extracted with solvent

Techniques Used: SDS Page, Mutagenesis, Sonication

12) Product Images from "The Membrane Localization Domain Is Required for Intracellular Localization and Autoregulation of YopE in Yersinia pseudotuberculosis ▿"

Article Title: The Membrane Localization Domain Is Required for Intracellular Localization and Autoregulation of YopE in Yersinia pseudotuberculosis ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.00333-09

(A) YopE is required for translocation control. HeLa cells were infected in duplicates with the indicated Yersinia strains for 3 h. Proteinase K was added to remove all extracellular proteins followed by lysis of one set of HeLa cells using digitonin.
Figure Legend Snippet: (A) YopE is required for translocation control. HeLa cells were infected in duplicates with the indicated Yersinia strains for 3 h. Proteinase K was added to remove all extracellular proteins followed by lysis of one set of HeLa cells using digitonin.

Techniques Used: Translocation Assay, Infection, Lysis

13) Product Images from "Different Behavior toward Bovine Spongiform Encephalopathy Infection of Bovine Prion Protein Transgenic Mice with One Extra Repeat Octapeptide Insert Mutation"

Article Title: Different Behavior toward Bovine Spongiform Encephalopathy Infection of Bovine Prion Protein Transgenic Mice with One Extra Repeat Octapeptide Insert Mutation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3811-03.2004

A , Comparative studies using brain homogenates from silent carriers as inoculum. Bo6ORTg and bo7ORTg mice were inoculated with bo6ORTgBSE 1 120d (120) and bo6ORTgBSE 1 150d (150) (two pools of brain homogenates from bo6ORTg mice killed at 120 or 150 d after inoculation with BSE 1 inoculum). Both inocula contained amounts of PrP res undetectable by conventional immunoblot analysis. Tg mice were killed at 330 d after inoculation, and PrP res from brain samples were analyzed by Western blot after proteinase K (20 μg/ml) treatment. Columns indicate the percentage of Tg mice showing PrP res at the indicated time after inoculation. B , Comparative kinetics of PrP res detection after BSE 2 inoculation in bo6ORTg and bo7ORTg mice. Bovine transgenic mice expressing 6OR-PrP or 7OR-PrP were inoculated with BSE 2 inoculum and killed at the indicated time points, irrespective of the onset of neurological signs. PrP res in brain samples were analyzed by Western blotting after proteinase K (20 μg/ml) treatment. Columns indicate the percentage of Tg mice showing PrP res at the indicated time after inoculation.
Figure Legend Snippet: A , Comparative studies using brain homogenates from silent carriers as inoculum. Bo6ORTg and bo7ORTg mice were inoculated with bo6ORTgBSE 1 120d (120) and bo6ORTgBSE 1 150d (150) (two pools of brain homogenates from bo6ORTg mice killed at 120 or 150 d after inoculation with BSE 1 inoculum). Both inocula contained amounts of PrP res undetectable by conventional immunoblot analysis. Tg mice were killed at 330 d after inoculation, and PrP res from brain samples were analyzed by Western blot after proteinase K (20 μg/ml) treatment. Columns indicate the percentage of Tg mice showing PrP res at the indicated time after inoculation. B , Comparative kinetics of PrP res detection after BSE 2 inoculation in bo6ORTg and bo7ORTg mice. Bovine transgenic mice expressing 6OR-PrP or 7OR-PrP were inoculated with BSE 2 inoculum and killed at the indicated time points, irrespective of the onset of neurological signs. PrP res in brain samples were analyzed by Western blotting after proteinase K (20 μg/ml) treatment. Columns indicate the percentage of Tg mice showing PrP res at the indicated time after inoculation.

Techniques Used: Mouse Assay, Western Blot, Transgenic Assay, Expressing

14) Product Images from "Detection of protease-resistant cervid prion protein in water from a CWD-endemic area"

Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

Journal: Prion

doi:

PrP CWD  amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD  from five replicate
Figure Legend Snippet: PrP CWD amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD from five replicate

Techniques Used: Amplification

Effects of flocculation and alum on PrP CWD  amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD  precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples
Figure Legend Snippet: Effects of flocculation and alum on PrP CWD amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples

Techniques Used: Flocculation, Amplification

PrP CWD  detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and
Figure Legend Snippet: PrP CWD detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and

Techniques Used: Amplification

PrP CWD  detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir
Figure Legend Snippet: PrP CWD detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir

Techniques Used: Amplification

Abrogation of PrP CWD  amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C  substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows
Figure Legend Snippet: Abrogation of PrP CWD amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows

Techniques Used: Amplification, Western Blot

Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons
Figure Legend Snippet: Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons

Techniques Used: Amplification, Molecular Weight

15) Product Images from "Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle"

Article Title: Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00119-15

Effects of PPD administration for skin tests on avidity indices for proteinase K-digested whole-cell sonicate of  M. bovis  (WCS-PK) antigens (A) or MPB83/MPB70 antigens (B) measured by ELISA. Sample collection time points were as follows: post CFT, 14
Figure Legend Snippet: Effects of PPD administration for skin tests on avidity indices for proteinase K-digested whole-cell sonicate of M. bovis (WCS-PK) antigens (A) or MPB83/MPB70 antigens (B) measured by ELISA. Sample collection time points were as follows: post CFT, 14

Techniques Used: Enzyme-linked Immunosorbent Assay

Kinetics of serum antibody responses to complex (WCS-PK) and specific (MPB83/MPB70) antigens and effects of skin tests, as measured by ELISA. (A) ELISA results for proteinase K-digested whole-cell sonicate (WCS-PK) of  M. bovis  over time after an  M. bovis
Figure Legend Snippet: Kinetics of serum antibody responses to complex (WCS-PK) and specific (MPB83/MPB70) antigens and effects of skin tests, as measured by ELISA. (A) ELISA results for proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis over time after an M. bovis

Techniques Used: Enzyme-linked Immunosorbent Assay

16) Product Images from "High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study"

Article Title: High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study

Journal: European Thyroid Journal

doi: 10.1159/000430092

Different clusters of HRM melting curves. The three different clusters, present in the picture, are obtained by the amplification and HRM analysis of exon 15 of the BRAF gene. While the DNA of V600D+ and V600E+ controls (green and red curves in a-d , respectively) were extracted by a commercial kit, the somatic DNA from wFNAB was obtained by pelletting the cells and by using 60 µl of lysis buffer (50 mM Tris-HCl at pH 8.5, 1 mM EDTA, 0.5% Tween 20 and sterile water) with 20 µl of proteinase K (10 mg/ml; Roche Diagnostics), and by incubating the samples at 56°C overnight and then at 95°C for 20 min to have a high yield from few cells also. The cluster of BRAF -samples is represented by a blue curve. All HRM analyses were performed using the SSO Fast Eva Green Supermix 2× (Bio-Rad Laboratories) and the same following protocol: 98°C for 2 min, 44 cycles of 3 s at 98°C and 30 s at 56.1°C, 1 cycle at 98°C for 30 s and 65°C for 1 min and 30 s, a progressive denaturation from 65 to 83°C, increasing the temperature by 0.2°C every 10 s and recording the fluorescence intensity for each increment. -d(RFU)/dT = Negative derivative (-d) of relative fluorescence units (RFU) over temperature (dT). a Original melting curves of V600D+, V600E+ and wild-type samples in green, red and blue, respectively. b Melting peaks showing the three characteristic shapes of V600D+ (green), V600E+ (red) and wild-type (blue) samples. c Normalization of V600D+ (green), V600E+ (red) and wild-type (blue) melting curves. d The differences between the three normalized curves, belonging to V600D+ (green), V600E+ (red) and wild-type (blue) samples.
Figure Legend Snippet: Different clusters of HRM melting curves. The three different clusters, present in the picture, are obtained by the amplification and HRM analysis of exon 15 of the BRAF gene. While the DNA of V600D+ and V600E+ controls (green and red curves in a-d , respectively) were extracted by a commercial kit, the somatic DNA from wFNAB was obtained by pelletting the cells and by using 60 µl of lysis buffer (50 mM Tris-HCl at pH 8.5, 1 mM EDTA, 0.5% Tween 20 and sterile water) with 20 µl of proteinase K (10 mg/ml; Roche Diagnostics), and by incubating the samples at 56°C overnight and then at 95°C for 20 min to have a high yield from few cells also. The cluster of BRAF -samples is represented by a blue curve. All HRM analyses were performed using the SSO Fast Eva Green Supermix 2× (Bio-Rad Laboratories) and the same following protocol: 98°C for 2 min, 44 cycles of 3 s at 98°C and 30 s at 56.1°C, 1 cycle at 98°C for 30 s and 65°C for 1 min and 30 s, a progressive denaturation from 65 to 83°C, increasing the temperature by 0.2°C every 10 s and recording the fluorescence intensity for each increment. -d(RFU)/dT = Negative derivative (-d) of relative fluorescence units (RFU) over temperature (dT). a Original melting curves of V600D+, V600E+ and wild-type samples in green, red and blue, respectively. b Melting peaks showing the three characteristic shapes of V600D+ (green), V600E+ (red) and wild-type (blue) samples. c Normalization of V600D+ (green), V600E+ (red) and wild-type (blue) melting curves. d The differences between the three normalized curves, belonging to V600D+ (green), V600E+ (red) and wild-type (blue) samples.

Techniques Used: Amplification, Lysis, Fluorescence

17) Product Images from "Distinct molecular phenotypes in bovine prion diseases"

Article Title: Distinct molecular phenotypes in bovine prion diseases

Journal: EMBO Reports

doi: 10.1038/sj.embor.7400054

( A ) Western blot detection of PrP res  from proteinase K-treated and ultracentrifuged brain homogenates, using RB1 polyclonal antibody (105–120 bovine epitope). Atypical cattle-BSE cases (A1 F  and A2 F ) (lanes 3 and 5) show, as do scrapie experimentally infected cattle (C Scr ) (lane 1), a higher molecular mass of the unglycosylated bands (arrow), compared to typical cattle-BSE cases (T UK , lane 2; T1 F , lane 4). Similar differences are also observed in sheep, between natural scrapie (SH NS ) (lane 7) and experimental BSE (SH BSE ) (lane 6). Brain equivalent quantities loaded per lane from lanes 1 to 7 are 2.4, 4.8, 38, 2.4, 38, 1.2 and 1.2 mg, respectively.  (B)  Mean molecular weights±standard deviations obtained for the di-, mono- and unglycosylated PrP res  bands (A: atypical cattle BSE; T: typical cattle BSE; F: French isolate; UK: British isolate; SH: sheep).
Figure Legend Snippet: ( A ) Western blot detection of PrP res from proteinase K-treated and ultracentrifuged brain homogenates, using RB1 polyclonal antibody (105–120 bovine epitope). Atypical cattle-BSE cases (A1 F and A2 F ) (lanes 3 and 5) show, as do scrapie experimentally infected cattle (C Scr ) (lane 1), a higher molecular mass of the unglycosylated bands (arrow), compared to typical cattle-BSE cases (T UK , lane 2; T1 F , lane 4). Similar differences are also observed in sheep, between natural scrapie (SH NS ) (lane 7) and experimental BSE (SH BSE ) (lane 6). Brain equivalent quantities loaded per lane from lanes 1 to 7 are 2.4, 4.8, 38, 2.4, 38, 1.2 and 1.2 mg, respectively. (B) Mean molecular weights±standard deviations obtained for the di-, mono- and unglycosylated PrP res bands (A: atypical cattle BSE; T: typical cattle BSE; F: French isolate; UK: British isolate; SH: sheep).

Techniques Used: Western Blot, Infection

18) Product Images from "A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins"

Article Title: A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

Journal: Genes & Development

doi: 10.1101/gad.1022002

Analysis of dFMR1 interaction with the ribosomal proteins L5 and L11. ( A ) Delineation of dFMR1 to determine the binding domains with L5 and L11. A fragment, dFLeu, containing the region equivalent to the ribosome-binding domain in hFMR1 interacted with neither L5 nor L11. A delineated fragment, dFC150, interacted with  35 S-labeled L5 and L11 as in the case of dFC181, whereas dFC120 did not, demonstrating that dFC150 is the L5 and L11 binding domain in dFMR1. ( B ) The binding domain to L5 and L11 confers to dFMR1 the activity of interacting with ribosomes. A truncated mutant of dFMR1 lacking the binding domains with L5 and L11 (dFMR1Δ150) was expressed in S2 cells and the cytoplasmic lysate was subjected to sedimentation on a linear density sucrose gradient. Western blots were performed on the fractions using anti-dFMR1 antibody. ( C ) Northern blot on TEV extracts obtained from cytoplasmic lysates with and without dFMR1-TAP. After protease K treatment of the TEV extracts, RNA molecules were recovered and resolved on a 10% denaturing gel containing 6 M urea. A Northern blot was then performed using a riboprobe specific for 5S rRNA. The total RNA lane contains the total RNA isolated from the parental S2 cells. Mass markers are indicated at  left . ( D ) dFMR1 is able to interact directly with L5 and L11 in vitro. GST pull-down assays were carried out using bacterially expressed His-L5 and His-L11, and Western blots with anti-His antibody were performed. ( E ) The ternary complex formation of 5S rRNA/L5/dFMR1 in vitro. 5S rRNA was labeled with [ 32 P]UTP. When dFMR1 was incubated with preformed L5/5S rRNA, the RNA band was super-shifted. The migration of free 5S rRNA, 5S rRNA plus BSA, or 5S rRNA plus L5 are shown.
Figure Legend Snippet: Analysis of dFMR1 interaction with the ribosomal proteins L5 and L11. ( A ) Delineation of dFMR1 to determine the binding domains with L5 and L11. A fragment, dFLeu, containing the region equivalent to the ribosome-binding domain in hFMR1 interacted with neither L5 nor L11. A delineated fragment, dFC150, interacted with 35 S-labeled L5 and L11 as in the case of dFC181, whereas dFC120 did not, demonstrating that dFC150 is the L5 and L11 binding domain in dFMR1. ( B ) The binding domain to L5 and L11 confers to dFMR1 the activity of interacting with ribosomes. A truncated mutant of dFMR1 lacking the binding domains with L5 and L11 (dFMR1Δ150) was expressed in S2 cells and the cytoplasmic lysate was subjected to sedimentation on a linear density sucrose gradient. Western blots were performed on the fractions using anti-dFMR1 antibody. ( C ) Northern blot on TEV extracts obtained from cytoplasmic lysates with and without dFMR1-TAP. After protease K treatment of the TEV extracts, RNA molecules were recovered and resolved on a 10% denaturing gel containing 6 M urea. A Northern blot was then performed using a riboprobe specific for 5S rRNA. The total RNA lane contains the total RNA isolated from the parental S2 cells. Mass markers are indicated at left . ( D ) dFMR1 is able to interact directly with L5 and L11 in vitro. GST pull-down assays were carried out using bacterially expressed His-L5 and His-L11, and Western blots with anti-His antibody were performed. ( E ) The ternary complex formation of 5S rRNA/L5/dFMR1 in vitro. 5S rRNA was labeled with [ 32 P]UTP. When dFMR1 was incubated with preformed L5/5S rRNA, the RNA band was super-shifted. The migration of free 5S rRNA, 5S rRNA plus BSA, or 5S rRNA plus L5 are shown.

Techniques Used: Binding Assay, Labeling, Activity Assay, Mutagenesis, Sedimentation, Western Blot, Northern Blot, Isolation, In Vitro, Incubation, Migration

19) Product Images from "A30P ?-Synuclein interferes with the stable integration of adult-born neurons into the olfactory network"

Article Title: A30P ?-Synuclein interferes with the stable integration of adult-born neurons into the olfactory network

Journal: Scientific Reports

doi: 10.1038/srep03931

Olfactory bulb pathology in A30P α-SYN mice. (a) Immunofluorescent micrographs showing the overexpression pattern of human A30P α-SYN (15G7, red) under control of the Thy1-promoter in the OB of 6-month-old transgenic mice: z-stack projections of confocal series. Scale bar - 50 μm. (b–e) Representative OB sections immunostained for phosphorylated α-SYN (pSer129, DAB), a marker for aberrant protein modification. Scale bars - 50 μm. (b) Distribution of phosphorylated α-SYN in (c) PGNs, (d) MCs and (e) GCs. Note that only MCs are positive for pSer129. (f–j) Representative OB sections with immunostaining for fibrillar protein, detected by Proteinase K digestion and human α-SYN specific antibody (15G7, DAB). Scale bars - 50 μm. Images with higher magnification of the (g) glomerular, (h) MC and (i) GC layer. Some α-SYN aggregates were detected in MCs of transgenic, but not wild-type, mice. (j) Olfactory discrimination task. Six-month-old Control (wild-type) ( n = 8) and A30P α-SYN mice ( n = 7) were exposed to different binary odour mixtures (e.g. 55% S + and 45% S − vs . 45% S + and 55% S − ). Mice were trained to dig in a bowl containing more of S + odour. Correct choices for S + were plotted against the different mixture pairs. Note that with increasing similarity of the mixtures, the performance of A30P α-SYN mice dropped compared to Control. (k) Olfactory memory task. ** p
Figure Legend Snippet: Olfactory bulb pathology in A30P α-SYN mice. (a) Immunofluorescent micrographs showing the overexpression pattern of human A30P α-SYN (15G7, red) under control of the Thy1-promoter in the OB of 6-month-old transgenic mice: z-stack projections of confocal series. Scale bar - 50 μm. (b–e) Representative OB sections immunostained for phosphorylated α-SYN (pSer129, DAB), a marker for aberrant protein modification. Scale bars - 50 μm. (b) Distribution of phosphorylated α-SYN in (c) PGNs, (d) MCs and (e) GCs. Note that only MCs are positive for pSer129. (f–j) Representative OB sections with immunostaining for fibrillar protein, detected by Proteinase K digestion and human α-SYN specific antibody (15G7, DAB). Scale bars - 50 μm. Images with higher magnification of the (g) glomerular, (h) MC and (i) GC layer. Some α-SYN aggregates were detected in MCs of transgenic, but not wild-type, mice. (j) Olfactory discrimination task. Six-month-old Control (wild-type) ( n = 8) and A30P α-SYN mice ( n = 7) were exposed to different binary odour mixtures (e.g. 55% S + and 45% S − vs . 45% S + and 55% S − ). Mice were trained to dig in a bowl containing more of S + odour. Correct choices for S + were plotted against the different mixture pairs. Note that with increasing similarity of the mixtures, the performance of A30P α-SYN mice dropped compared to Control. (k) Olfactory memory task. ** p

Techniques Used: Mouse Assay, Over Expression, Transgenic Assay, Marker, Modification, Immunostaining

20) Product Images from "Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils"

Article Title: Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105433

Amyloid fibrils maintained their amyloid architecture after proteolytic digestion and acetone extraction. (A) Aβ 1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar (upper gel) or soluble (lower gel) state were incubated in the absence or presence of 0.13 µg/ml (1∶500, w/w) proteinase K (PK) for 2 h at 42°C. The digestion was conducted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl buffer. The reaction was stopped by boiling the samples in Laemmli buffer with 2% SDS and the samples were resolved by 16% SDS-PAGE. Western blot using 6E10 (Aβ 1–40 ), syn-1 (α-syn) or a gelsolin-specific antibody is presented. (B) The same reaction described in panel A was performed in the presence of 20 µM of thioflavin T (ThT) and the florescence was monitored every 10 min. Ex = 440 nm and Em = 485 nm. (C) Aβ 1–40 amyloid fibrils at 65 µg/ml concentration were diluted in 1 volume (1 V) of PBS, hexane, acetone or chloroform and centrifuged (16,000 g) for 10 min at 4°C. The pellet was resuspended in phosphate buffer with 20 µM ThT and the fluorescence measured. An aliquot of undiluted/uncentrifuged fibrils was used as the load. Ex = 450 nm and Em = 465–520 nm.
Figure Legend Snippet: Amyloid fibrils maintained their amyloid architecture after proteolytic digestion and acetone extraction. (A) Aβ 1–40, α-syn or gelsolin peptides (65 µg/ml) in a fibrillar (upper gel) or soluble (lower gel) state were incubated in the absence or presence of 0.13 µg/ml (1∶500, w/w) proteinase K (PK) for 2 h at 42°C. The digestion was conducted in 50 mM sodium phosphate, pH 7.4, 150 mM NaCl buffer. The reaction was stopped by boiling the samples in Laemmli buffer with 2% SDS and the samples were resolved by 16% SDS-PAGE. Western blot using 6E10 (Aβ 1–40 ), syn-1 (α-syn) or a gelsolin-specific antibody is presented. (B) The same reaction described in panel A was performed in the presence of 20 µM of thioflavin T (ThT) and the florescence was monitored every 10 min. Ex = 440 nm and Em = 485 nm. (C) Aβ 1–40 amyloid fibrils at 65 µg/ml concentration were diluted in 1 volume (1 V) of PBS, hexane, acetone or chloroform and centrifuged (16,000 g) for 10 min at 4°C. The pellet was resuspended in phosphate buffer with 20 µM ThT and the fluorescence measured. An aliquot of undiluted/uncentrifuged fibrils was used as the load. Ex = 450 nm and Em = 465–520 nm.

Techniques Used: Incubation, SDS Page, Western Blot, Concentration Assay, Fluorescence

Effect of proteinase K digestion and acetone precipitation on the protein content of a complex biological extract. (A and B) The complex biological extract was obtained by mechanical disruption of wild type C. elegans worms followed by a brief centrifugation (700 g for 3 min) to remove unlysed worms. Aβ 1–40 fibrils (0.2% w/w protein concentration) were added to the worm post debris supernatant (PDS) and the samples were digested with PK (1∶500) for 2 h at 42°C followed by acetone precipitation. An aliquot before PK digestion (load), after PK digestion (+ PK) and after PK digestion and acetone precipitation (+ PK/acetone) were resolved by SDS-PAGE (A) or the protein was quantified by BCA assay (B). In the panel A, the upper gel is silver stained and the lower gel is a Western blot for Aβ using the 6E10 antibody. (C–F) TEM images of PDS. PDS was incubated in the absence (C) or in the presence of 0.2% Aβ 1–40 fibrils (E) before the PK/acetone step. PDS incubated in the absence (D) or in the presence of 0.2% Aβ 1–40 fibrils (F) was digested with PK and precipitated with acetone. Note that amyloid fibrils are present only in the samples to which Aβ 1–40 fibrils were added (E and F).
Figure Legend Snippet: Effect of proteinase K digestion and acetone precipitation on the protein content of a complex biological extract. (A and B) The complex biological extract was obtained by mechanical disruption of wild type C. elegans worms followed by a brief centrifugation (700 g for 3 min) to remove unlysed worms. Aβ 1–40 fibrils (0.2% w/w protein concentration) were added to the worm post debris supernatant (PDS) and the samples were digested with PK (1∶500) for 2 h at 42°C followed by acetone precipitation. An aliquot before PK digestion (load), after PK digestion (+ PK) and after PK digestion and acetone precipitation (+ PK/acetone) were resolved by SDS-PAGE (A) or the protein was quantified by BCA assay (B). In the panel A, the upper gel is silver stained and the lower gel is a Western blot for Aβ using the 6E10 antibody. (C–F) TEM images of PDS. PDS was incubated in the absence (C) or in the presence of 0.2% Aβ 1–40 fibrils (E) before the PK/acetone step. PDS incubated in the absence (D) or in the presence of 0.2% Aβ 1–40 fibrils (F) was digested with PK and precipitated with acetone. Note that amyloid fibrils are present only in the samples to which Aβ 1–40 fibrils were added (E and F).

Techniques Used: Centrifugation, Protein Concentration, SDS Page, BIA-KA, Staining, Western Blot, Transmission Electron Microscopy, Incubation

21) Product Images from "Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation"

Article Title: Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20062032

A method to detect ssDNA in the context of chromatin.  (A) Two assays were used to detect ssDNA, one using purified genomic DNA (Deproteinized DNA) (reference   18 ) and another using DNA in fixed nuclei (Chromatinized DNA). Both kinds of substrates were treated with bisulfite, amplified, cloned, and sequenced as described in Materials and methods. (B–D) Frequency of C to T conversions (indicative of ssDNA on the noncoding strand) and G to A conversions (indicating ssDNA on the template strand) was measured for untreated DNA (B), deproteinized DNA (C), and chromatinized DNA (D) treated with bisulfite in the  IgH V  region of Ramos cells (depicted above the diagrams). L indicates the leader sequence. Position 273 is a hotspot for G to A mutations. (E) Examples of ssDNA patches on the coding and noncoding strands. Asterisks indicate bases that have been converted by bisulfite. Because the length of these patches cannot be determined exactly, the minimum size is shown as boxes, whereas the maximum size is shown as a horizontal line. (F) The frequency of ssDNA patches was calculated for  IgH V  region in chromatinized DNA from Ramos 7 cells untreated (control) or pretreated with proteinase K. Numbers above the bar graphs indicate p-values calculated using the Chi-squared test. The results are representative of three experiments. Sample sizes are indicated in Table S2.
Figure Legend Snippet: A method to detect ssDNA in the context of chromatin. (A) Two assays were used to detect ssDNA, one using purified genomic DNA (Deproteinized DNA) (reference 18 ) and another using DNA in fixed nuclei (Chromatinized DNA). Both kinds of substrates were treated with bisulfite, amplified, cloned, and sequenced as described in Materials and methods. (B–D) Frequency of C to T conversions (indicative of ssDNA on the noncoding strand) and G to A conversions (indicating ssDNA on the template strand) was measured for untreated DNA (B), deproteinized DNA (C), and chromatinized DNA (D) treated with bisulfite in the IgH V region of Ramos cells (depicted above the diagrams). L indicates the leader sequence. Position 273 is a hotspot for G to A mutations. (E) Examples of ssDNA patches on the coding and noncoding strands. Asterisks indicate bases that have been converted by bisulfite. Because the length of these patches cannot be determined exactly, the minimum size is shown as boxes, whereas the maximum size is shown as a horizontal line. (F) The frequency of ssDNA patches was calculated for IgH V region in chromatinized DNA from Ramos 7 cells untreated (control) or pretreated with proteinase K. Numbers above the bar graphs indicate p-values calculated using the Chi-squared test. The results are representative of three experiments. Sample sizes are indicated in Table S2.

Techniques Used: Purification, Amplification, Clone Assay, Sequencing

22) Product Images from "Accumulation of Pathological Prion Protein PrPSc in the Skin of Animals with Experimental and Natural Scrapie"

Article Title: Accumulation of Pathological Prion Protein PrPSc in the Skin of Animals with Experimental and Natural Scrapie

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0030066

Time-Course of PrP Sc  Deposition in Skin Tissue (A–E) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , extracted from different skin samples of hamsters orally challenged with 263K scrapie and sacrificed at the following time-points after infection: (A) 70 dpi, (B) 100 dpi, (C) 130 dpi, (D) at the onset of clinical signs for scrapie (138–146 dpi), and (E) at the terminal stage of disease (157–171 dpi). Lanes with test samples: S1, skin sample from hindlimb; S2, skin sample from forelimb; S3, skin sample from back; S4, skin sample from abdomen; S5, skin sample from head. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill 263K scrapie hamsters containing 1 × 10 −7  g brain tissue. Representative results are shown for each stage of incubation. Substantial individual variation was observed at 130 dpi, with two of five and three of five animals displaying findings as in (C) in the Western blot on the left-hand side or the Western blot on the right-hand side, respectively. (F) Lanes S1d–S5d: Same samples as in S1–S5 of (E) but deglycosylated with PNGaseF. (A–F) Amounts of tissue represented in lanes: (A) S1, 43 mg; S2, 52 mg; S3, 68 mg; S4, 58 mg; S5, 73 mg; (B) S1, 78 mg; S2, 44 mg; S3, 63 mg; S4, 67 mg; S5, 50 mg; ([C], Western blot on the left side) S1, 42 mg; S2, 76 mg; S3, 61 mg; S4, 58 mg; S5, 73 mg; ([C], Western blot on the right side) S1, 51 mg; S2, 63 mg; S3, 70 mg; S4, 87 mg; S5, 54 mg; (D) S1, 63 mg; S2, 68 mg; S3, 90 mg; S4, 50 mg; S5, 68 mg; (E) S1, 55 mg; S2, 73 mg; S3, 80 mg; S4, 88 mg; S5, 70 mg; (F) S1d, 12 mg; S2d, 14 mg; S3d, 19 mg; S4d, 12 mg; S5d, 20 mg. (G) Time-scale displaying the mean incubation period and the pre-clinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time-points at which animals were tested for PrP Sc  in skin samples.
Figure Legend Snippet: Time-Course of PrP Sc Deposition in Skin Tissue (A–E) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , extracted from different skin samples of hamsters orally challenged with 263K scrapie and sacrificed at the following time-points after infection: (A) 70 dpi, (B) 100 dpi, (C) 130 dpi, (D) at the onset of clinical signs for scrapie (138–146 dpi), and (E) at the terminal stage of disease (157–171 dpi). Lanes with test samples: S1, skin sample from hindlimb; S2, skin sample from forelimb; S3, skin sample from back; S4, skin sample from abdomen; S5, skin sample from head. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill 263K scrapie hamsters containing 1 × 10 −7 g brain tissue. Representative results are shown for each stage of incubation. Substantial individual variation was observed at 130 dpi, with two of five and three of five animals displaying findings as in (C) in the Western blot on the left-hand side or the Western blot on the right-hand side, respectively. (F) Lanes S1d–S5d: Same samples as in S1–S5 of (E) but deglycosylated with PNGaseF. (A–F) Amounts of tissue represented in lanes: (A) S1, 43 mg; S2, 52 mg; S3, 68 mg; S4, 58 mg; S5, 73 mg; (B) S1, 78 mg; S2, 44 mg; S3, 63 mg; S4, 67 mg; S5, 50 mg; ([C], Western blot on the left side) S1, 42 mg; S2, 76 mg; S3, 61 mg; S4, 58 mg; S5, 73 mg; ([C], Western blot on the right side) S1, 51 mg; S2, 63 mg; S3, 70 mg; S4, 87 mg; S5, 54 mg; (D) S1, 63 mg; S2, 68 mg; S3, 90 mg; S4, 50 mg; S5, 68 mg; (E) S1, 55 mg; S2, 73 mg; S3, 80 mg; S4, 88 mg; S5, 70 mg; (F) S1d, 12 mg; S2d, 14 mg; S3d, 19 mg; S4d, 12 mg; S5d, 20 mg. (G) Time-scale displaying the mean incubation period and the pre-clinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time-points at which animals were tested for PrP Sc in skin samples.

Techniques Used: Western Blot, Infection, Incubation

PrP Sc  Routing to the Skin and to Components of the Lymphoreticular System of Hamsters Challenged via Different Routes with 263K Scrapie Agent (A) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , in skin specimens from terminally ill scrapie hamsters. Lanes 1, 2, and 3: skin samples from orally mock-infected control hamsters, spiked before extraction with 1 × 10 −6  g, 5 × 10 −6  g, or 1 × 10 −5  g of brain homogenate from terminally ill 263K hamsters. Lanes 4 and 5: skin samples from hindlimbs and forelimbs of hamsters orally infected with scrapie brain homogenate. Lanes 6 and 7: skin samples from hindlimbs and forelimbs of hamsters intracerebrally infected with scrapie brain homogenate. Lanes 8 and 9: skin samples from hindlimbs and forelimbs of hamsters infected by implantation of s.w. contaminated with scrapie agent. Lanes 10 and 11: skin samples from hindlimbs and forelimbs of hamsters infected peripherally by f.p. inoculation of scrapie brain homogenate. Lanes 12 and 13: skin samples from hindlimbs and forelimbs of hamsters orally mock-infected with normal brain homogenate. Amounts of tissue represented in lanes: 1, 53mg; 2, 58 mg; 3, 68 mg; 4, 68 mg; 5, 75 mg; 6, 78 mg; 7, 64 mg; 8, 69 mg; 9, 60 mg; 10, 62 mg; 11, 73 mg; 12, 61 mg; 13, 58 mg. (B) Western blot detection of PrP27–30 in spleens and selected lymph nodes from terminally ill scrapie hamsters. Lanes 1 and 5: proteinase K-digested brain homogenate from terminally ill scrapie hamsters, containing 1 × 10 −7  g brain tissue. Lanes 2–4: spleen samples from p.o.- (2), s.w.-, (3) and i.c.-infected (4) hamsters. Lanes 6–8: mesenteric lymph node samples from p.o.- (6), s.w.-, (7) and i.c.-infected (8) hamsters. Amounts of tissue represented in lanes: 2, 40 mg; 3, 45 mg; 4, 41 mg; 6, 6 mg; 7, 8 mg; 8, 6 mg.
Figure Legend Snippet: PrP Sc Routing to the Skin and to Components of the Lymphoreticular System of Hamsters Challenged via Different Routes with 263K Scrapie Agent (A) Western blot detection of PrP27–30, the protease-resistant core of PrP Sc , in skin specimens from terminally ill scrapie hamsters. Lanes 1, 2, and 3: skin samples from orally mock-infected control hamsters, spiked before extraction with 1 × 10 −6 g, 5 × 10 −6 g, or 1 × 10 −5 g of brain homogenate from terminally ill 263K hamsters. Lanes 4 and 5: skin samples from hindlimbs and forelimbs of hamsters orally infected with scrapie brain homogenate. Lanes 6 and 7: skin samples from hindlimbs and forelimbs of hamsters intracerebrally infected with scrapie brain homogenate. Lanes 8 and 9: skin samples from hindlimbs and forelimbs of hamsters infected by implantation of s.w. contaminated with scrapie agent. Lanes 10 and 11: skin samples from hindlimbs and forelimbs of hamsters infected peripherally by f.p. inoculation of scrapie brain homogenate. Lanes 12 and 13: skin samples from hindlimbs and forelimbs of hamsters orally mock-infected with normal brain homogenate. Amounts of tissue represented in lanes: 1, 53mg; 2, 58 mg; 3, 68 mg; 4, 68 mg; 5, 75 mg; 6, 78 mg; 7, 64 mg; 8, 69 mg; 9, 60 mg; 10, 62 mg; 11, 73 mg; 12, 61 mg; 13, 58 mg. (B) Western blot detection of PrP27–30 in spleens and selected lymph nodes from terminally ill scrapie hamsters. Lanes 1 and 5: proteinase K-digested brain homogenate from terminally ill scrapie hamsters, containing 1 × 10 −7 g brain tissue. Lanes 2–4: spleen samples from p.o.- (2), s.w.-, (3) and i.c.-infected (4) hamsters. Lanes 6–8: mesenteric lymph node samples from p.o.- (6), s.w.-, (7) and i.c.-infected (8) hamsters. Amounts of tissue represented in lanes: 2, 40 mg; 3, 45 mg; 4, 41 mg; 6, 6 mg; 7, 8 mg; 8, 6 mg.

Techniques Used: Western Blot, Infection

23) Product Images from "RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome"

Article Title: RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

Journal: Genome Biology

doi: 10.1186/gb-2014-15-1-r3

Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.
Figure Legend Snippet: Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.

Techniques Used: Next-Generation Sequencing, Sequencing, Expressing

24) Product Images from "Targeting Insulin Receptor with a Novel Internalizing Aptamer"

Article Title: Targeting Insulin Receptor with a Novel Internalizing Aptamer

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2016.73

Cell-internalizing SELEX. ( a ) Scheme of the cell-internalizing protocol. We performed two cycles in which the G13 pool was incubated with U87MG target cells. Unbound aptamers were discarded and cells were treated with proteinase K for 30 minutes to remove cell surface-bound aptamers. Internalized aptamers were recovered by RNA extraction and RT-PCR. ( b ) Dendrogram by MAFFT analysis of the 50 individual sequences cloned after the two rounds of internalization. The most represented sequences are boxed. ( c ) Scattering plot of the 100 most abundant aptamers obtained by Illumina deep sequence analyses of the two cycles of internalization. The top five aptamers are indicated. SELEX, Systematic Evolution of Ligands by Exponential enrichment; RT-PCR, reverse transcription polymerase chain reaction.
Figure Legend Snippet: Cell-internalizing SELEX. ( a ) Scheme of the cell-internalizing protocol. We performed two cycles in which the G13 pool was incubated with U87MG target cells. Unbound aptamers were discarded and cells were treated with proteinase K for 30 minutes to remove cell surface-bound aptamers. Internalized aptamers were recovered by RNA extraction and RT-PCR. ( b ) Dendrogram by MAFFT analysis of the 50 individual sequences cloned after the two rounds of internalization. The most represented sequences are boxed. ( c ) Scattering plot of the 100 most abundant aptamers obtained by Illumina deep sequence analyses of the two cycles of internalization. The top five aptamers are indicated. SELEX, Systematic Evolution of Ligands by Exponential enrichment; RT-PCR, reverse transcription polymerase chain reaction.

Techniques Used: Incubation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing

25) Product Images from "Resistance of Capnocytophaga canimorsus to Killing by Human Complement and Polymorphonuclear Leukocytes ▿"

Article Title: Resistance of Capnocytophaga canimorsus to Killing by Human Complement and Polymorphonuclear Leukocytes ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.01324-08

Y1C12 has an altered lipidated polysaccharide structure. (A) Immunoblotting analysis of proteinase K-treated Cc5, Y1C12, and cY1C12 bacteria and of LPS isolated from Cc5 and Y1C12 using anti-Cc5. (B) Immunoblotting analysis as described above (A) using
Figure Legend Snippet: Y1C12 has an altered lipidated polysaccharide structure. (A) Immunoblotting analysis of proteinase K-treated Cc5, Y1C12, and cY1C12 bacteria and of LPS isolated from Cc5 and Y1C12 using anti-Cc5. (B) Immunoblotting analysis as described above (A) using

Techniques Used: Isolation

26) Product Images from "M-Ficolin Binds Selectively to the Capsular Polysaccharides of Streptococcus pneumoniae Serotypes 19B and 19C and of a Streptococcus mitis Strain"

Article Title: M-Ficolin Binds Selectively to the Capsular Polysaccharides of Streptococcus pneumoniae Serotypes 19B and 19C and of a Streptococcus mitis Strain

Journal: Infection and Immunity

doi: 10.1128/IAI.01148-12

Inhibition of binding of M-ficolin to  S. pneumoniae  strains of serotypes 19B and 19C. (A) The binding of serum M-ficolin to 19B and 19C strains (in the presence of 100 mM GlcNac or 100 mM glucose) and to proteinase K-treated bacteria was tested as described
Figure Legend Snippet: Inhibition of binding of M-ficolin to S. pneumoniae strains of serotypes 19B and 19C. (A) The binding of serum M-ficolin to 19B and 19C strains (in the presence of 100 mM GlcNac or 100 mM glucose) and to proteinase K-treated bacteria was tested as described

Techniques Used: Inhibition, Binding Assay

27) Product Images from "Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype"

Article Title: Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype

Journal: Journal of Virology

doi: 10.1128/JVI.01586-15

Antigenic mapping of PrP Sc from TME and CWD infection of transgenic mice and Syrian golden hamsters. (A) Prion-infected brain homogenates from HPrP7752KO mice and Syrian golden hamsters were digested with or without proteinase K (PK) and analyzed by SDS-PAGE
Figure Legend Snippet: Antigenic mapping of PrP Sc from TME and CWD infection of transgenic mice and Syrian golden hamsters. (A) Prion-infected brain homogenates from HPrP7752KO mice and Syrian golden hamsters were digested with or without proteinase K (PK) and analyzed by SDS-PAGE

Techniques Used: Infection, Transgenic Assay, Mouse Assay, SDS Page

Enrichment and deglycosylation of PrP Sc from TME and CWD strains. Brain homogenates from SGH infected with HY TME, DY TME, WST CWD, and CKY CWD were enriched for PrP Sc by detergent extraction, ultracentrifugation, and proteinase K digestion (A), and N-linked
Figure Legend Snippet: Enrichment and deglycosylation of PrP Sc from TME and CWD strains. Brain homogenates from SGH infected with HY TME, DY TME, WST CWD, and CKY CWD were enriched for PrP Sc by detergent extraction, ultracentrifugation, and proteinase K digestion (A), and N-linked

Techniques Used: Infection

28) Product Images from "Incongruity between Prion Conversion and Incubation Period following Coinfection"

Article Title: Incongruity between Prion Conversion and Incubation Period following Coinfection

Journal: Journal of Virology

doi: 10.1128/JVI.00409-16

Western blot detection of PrP Sc  from HY TME- and 139H-coinfected hamsters. Brain homogenates from hamsters infected with either the hyper (HY) 139H or drowsy (DY) TME strain at terminal disease were digested with proteinase K, and the unglycosylated PrP
Figure Legend Snippet: Western blot detection of PrP Sc from HY TME- and 139H-coinfected hamsters. Brain homogenates from hamsters infected with either the hyper (HY) 139H or drowsy (DY) TME strain at terminal disease were digested with proteinase K, and the unglycosylated PrP

Techniques Used: Western Blot, Infection

29) Product Images from "Copsin, a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis *"

Article Title: Copsin, a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.599878

Identification of an AMP in the secretome of  C. cinerea . A , proteins were extracted from the unchallenged  C. cinerea  secretome, digested with proteinase K, and the remaining proteins were fractionated on a cation exchange column. The effluent was monitored
Figure Legend Snippet: Identification of an AMP in the secretome of C. cinerea . A , proteins were extracted from the unchallenged C. cinerea secretome, digested with proteinase K, and the remaining proteins were fractionated on a cation exchange column. The effluent was monitored

Techniques Used:

30) Product Images from "GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis"

Article Title: GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis

Journal: Autophagy

doi: 10.4161/auto.7.12.18424

The GFP-Atg8 protease protection assay. Cultures of vam3 ts and atg1Δ cells expressing GFP-Atg8 grown to mid-log phase at 25°C were preincubated at 37°C for 30 min, and then shifted to starvation temperature at 37°C, for 1 h. Osmotically lysed cell extracts were analyzed for sensitivity to proteinase K (PK) in the presence or absence of 0.2% Triton X-100 (TX).
Figure Legend Snippet: The GFP-Atg8 protease protection assay. Cultures of vam3 ts and atg1Δ cells expressing GFP-Atg8 grown to mid-log phase at 25°C were preincubated at 37°C for 30 min, and then shifted to starvation temperature at 37°C, for 1 h. Osmotically lysed cell extracts were analyzed for sensitivity to proteinase K (PK) in the presence or absence of 0.2% Triton X-100 (TX).

Techniques Used: Expressing

31) Product Images from "Enzyme-labeled Antigen Method: Histochemical Detection of Antigen-specific Antibody-producing Cells in Tissue Sections of Rats Immunized With Horseradish Peroxidase, Ovalbumin, or Keyhole Limpet Hemocyanin"

Article Title: Enzyme-labeled Antigen Method: Histochemical Detection of Antigen-specific Antibody-producing Cells in Tissue Sections of Rats Immunized With Horseradish Peroxidase, Ovalbumin, or Keyhole Limpet Hemocyanin

Journal:

doi: 10.1369/jhc.2008.952259

( A ) Buffered formalin-fixed, paraffin-embedded sections of the axillary lymph nodes of HRP- or KLH-immunized rats reacted with HRP or biotinylated KLH after strong proteinase K pretreatment. A small number of plasma cells producing anti-HRP antibodies
Figure Legend Snippet: ( A ) Buffered formalin-fixed, paraffin-embedded sections of the axillary lymph nodes of HRP- or KLH-immunized rats reacted with HRP or biotinylated KLH after strong proteinase K pretreatment. A small number of plasma cells producing anti-HRP antibodies

Techniques Used: Formalin-fixed Paraffin-Embedded

32) Product Images from "Functional domain organization of the potato ?-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants"

Article Title: Functional domain organization of the potato ?-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants

Journal: Biochemical Journal

doi: 10.1042/BJ20041119

Coomassie Brilliant Blue-stained SDS/PAGE gel of the GWD generated proteolytic fragments GWD was digested with proteinase K at an enzyme to protease molar ratio of 200:1. At the indicated time points, an aliquot was taken and the digestion terminated by the addition of PMSF and 8 μg of total protein was loaded per lane. Std, molecular-mass standards (sizes given in kDa).
Figure Legend Snippet: Coomassie Brilliant Blue-stained SDS/PAGE gel of the GWD generated proteolytic fragments GWD was digested with proteinase K at an enzyme to protease molar ratio of 200:1. At the indicated time points, an aliquot was taken and the digestion terminated by the addition of PMSF and 8 μg of total protein was loaded per lane. Std, molecular-mass standards (sizes given in kDa).

Techniques Used: Staining, SDS Page, Generated

33) Product Images from "Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation"

Article Title: Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03452-15

Effects of enzyme treatment on the biofilms produced by  Capnocytophaga ochracea . (A) Total biomass of biofilms. Biofilms were grown for 48 h at 37°C under anaerobic conditions, treated with 0.5 mg/ml proteinase K, DNase I, or sodium metaperiodate
Figure Legend Snippet: Effects of enzyme treatment on the biofilms produced by Capnocytophaga ochracea . (A) Total biomass of biofilms. Biofilms were grown for 48 h at 37°C under anaerobic conditions, treated with 0.5 mg/ml proteinase K, DNase I, or sodium metaperiodate

Techniques Used: Produced

34) Product Images from "SYM1 Is the Stress-Induced Saccharomyces cerevisiae Ortholog of the Mammalian Kidney Disease Gene Mpv17 and Is Required for Ethanol Metabolism and Tolerance during Heat Shock"

Article Title: SYM1 Is the Stress-Induced Saccharomyces cerevisiae Ortholog of the Mammalian Kidney Disease Gene Mpv17 and Is Required for Ethanol Metabolism and Tolerance during Heat Shock

Journal: Eukaryotic Cell

doi: 10.1128/EC.3.3.620-631.2004

Sym1 is an integral membrane protein of the mitochondrial inner membrane. (A) sym1 Δ cells expressing the Sym1-HA fusion were grown in SC-URA, harvested, spheroplasted, and lysed by Dounce homogenization. The sample was split into two equal aliquots, one of which was held as the total cell extract (T); the other was subjected to centrifugation at 16,100 × g , and both supernatant (S) and membrane pellet (P) fractions were isolated. Equivalent amounts of all fractions were subjected to SDS-PAGE and Western blotting to identify the indicated proteins. (B) sym1 Δ cells expressing the Sym1-HA fusion were grown in SC-URA, harvested, and lysed using glass beads. A medium-speed membrane pellet was generated by centrifugation at 16,100 × g . The membrane pellet was split into thirds and treated with buffer, 0.1 M NaCO 3 , or 1% Triton X-100, and the aliquots were again centrifuged at 16,100 × g to isolate membrane pellet (P) and supernatant (S) fractions. SDS-PAGE and Western blotting was carried out as described for panel A. (C) Mitochondria were isolated for the protease protection assay as described in Materials and Methods. Mitochondria were split into equal aliquots and diluted into osmotically supportive buffer (mitochondria) or nonsupportive buffer (mitoplasts), which selectively ruptures the outer membrane. These samples were further diluted into three equivalent aliquots and treated with buffer, proteinase K (100 μg/ml), or proteinase K-1% Triton X-100. Equivalent amounts of all samples were resolved with SDS-PAGE and subjected to Western blotting with antisera as described previously. Fis1 was used as an outer membrane protein control.
Figure Legend Snippet: Sym1 is an integral membrane protein of the mitochondrial inner membrane. (A) sym1 Δ cells expressing the Sym1-HA fusion were grown in SC-URA, harvested, spheroplasted, and lysed by Dounce homogenization. The sample was split into two equal aliquots, one of which was held as the total cell extract (T); the other was subjected to centrifugation at 16,100 × g , and both supernatant (S) and membrane pellet (P) fractions were isolated. Equivalent amounts of all fractions were subjected to SDS-PAGE and Western blotting to identify the indicated proteins. (B) sym1 Δ cells expressing the Sym1-HA fusion were grown in SC-URA, harvested, and lysed using glass beads. A medium-speed membrane pellet was generated by centrifugation at 16,100 × g . The membrane pellet was split into thirds and treated with buffer, 0.1 M NaCO 3 , or 1% Triton X-100, and the aliquots were again centrifuged at 16,100 × g to isolate membrane pellet (P) and supernatant (S) fractions. SDS-PAGE and Western blotting was carried out as described for panel A. (C) Mitochondria were isolated for the protease protection assay as described in Materials and Methods. Mitochondria were split into equal aliquots and diluted into osmotically supportive buffer (mitochondria) or nonsupportive buffer (mitoplasts), which selectively ruptures the outer membrane. These samples were further diluted into three equivalent aliquots and treated with buffer, proteinase K (100 μg/ml), or proteinase K-1% Triton X-100. Equivalent amounts of all samples were resolved with SDS-PAGE and subjected to Western blotting with antisera as described previously. Fis1 was used as an outer membrane protein control.

Techniques Used: Expressing, Homogenization, Centrifugation, Isolation, SDS Page, Western Blot, Generated

35) Product Images from "Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein"

Article Title: Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein

Journal: The EMBO Journal

doi: 10.1093/emboj/20.19.5383

Fig. 5. Insoluble PrP in ALLN-treated cells is partially protease resistant. N2a-C10 cells were treated for 12 h with 150 µM ALLN. In ( A ), cell lysates were incubated with proteinase K (10 µg/ml, 37°C, 30 min, lanes 2 and 4) before western analysis with 3F4. A protease-resistant PrP species with a molecular weight of 19 kDa appeared in ALLN-treated cells (lane 4). In lane 5, the protease-treated lysate of prion-infected ScN2a-C10 cells was added for size comparison. The protease-resistant band in lane 4 co-migrated with the lower (unglycosylated) glycoform of PrP27–30 (lane 5). ( B ) The lysates of N2a-C10 cells were separated into high speed supernatants and pellets before proteolysis. The protease-resistant species were insoluble in Sarkosyl (lane 8). Proteolysis appeared to partially convert the 26 kDa aggregate into the 19 kDa band.
Figure Legend Snippet: Fig. 5. Insoluble PrP in ALLN-treated cells is partially protease resistant. N2a-C10 cells were treated for 12 h with 150 µM ALLN. In ( A ), cell lysates were incubated with proteinase K (10 µg/ml, 37°C, 30 min, lanes 2 and 4) before western analysis with 3F4. A protease-resistant PrP species with a molecular weight of 19 kDa appeared in ALLN-treated cells (lane 4). In lane 5, the protease-treated lysate of prion-infected ScN2a-C10 cells was added for size comparison. The protease-resistant band in lane 4 co-migrated with the lower (unglycosylated) glycoform of PrP27–30 (lane 5). ( B ) The lysates of N2a-C10 cells were separated into high speed supernatants and pellets before proteolysis. The protease-resistant species were insoluble in Sarkosyl (lane 8). Proteolysis appeared to partially convert the 26 kDa aggregate into the 19 kDa band.

Techniques Used: Incubation, Western Blot, Molecular Weight, Infection

36) Product Images from "Distinct types of translation termination generate substrates for ribosome-associated quality control"

Article Title: Distinct types of translation termination generate substrates for ribosome-associated quality control

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw566

Enrichment of elongator tRNAs in 60S fractions in Cdc48-depleted cells depends on Dom34. We treated individual ribosome-containing sucrose gradient fractions with proteinase K to separate tRNAs from the conjugated peptides. Next, the extracted RNAs were sequentially analyzed by northern hybridization of the same membrane with probes that detect tRNA Met_i , tRNA Glu , tRNA Leu , tRNA Val , tRNA Thr . The 18S and 25S rRNAs were analyzed on all membranes to verify alignment of the gradient fractions. ( A ) A representative example of the distribution of rRNA and tRNAs in gradient fractions is shown; see Supplementary Figure S4 for full hybridization sets. ( B ) The percentage of tRNAs in each fraction relative to the total was determined using phosphorimager quantification of the hybridization signals. The floating bars represent the full range of values obtained in each fraction (min to max) and the crossing lines indicate the mean. Four elongator tRNAs (top) or tRNA Met_i (bottom) were quantified in gradients prepared from 2 biological replicates of wild-type cells, 3 replicates of P TET-O7 -CDC48 and three replicates of dom34Δ P TET-O7 -CDC48 cells. ( C ) RNA in ribosomes pelleted from the 60S gradient fractions of the indicated strains was resolved on an acid-urea polyacrilamide gel and analyzed by northern hybridization using a radioactively labeled probe against tRNA Leu . Treatment with phenol (‘+’) was used to selectively remove long peptidyl-tRNAs. Prior to hybridization, the membrane was stained with methylene blue (MB) to control loading by visualizing 5S and 5.8S rRNAs in the 60S subunits. XC, the xylene cyanol band.
Figure Legend Snippet: Enrichment of elongator tRNAs in 60S fractions in Cdc48-depleted cells depends on Dom34. We treated individual ribosome-containing sucrose gradient fractions with proteinase K to separate tRNAs from the conjugated peptides. Next, the extracted RNAs were sequentially analyzed by northern hybridization of the same membrane with probes that detect tRNA Met_i , tRNA Glu , tRNA Leu , tRNA Val , tRNA Thr . The 18S and 25S rRNAs were analyzed on all membranes to verify alignment of the gradient fractions. ( A ) A representative example of the distribution of rRNA and tRNAs in gradient fractions is shown; see Supplementary Figure S4 for full hybridization sets. ( B ) The percentage of tRNAs in each fraction relative to the total was determined using phosphorimager quantification of the hybridization signals. The floating bars represent the full range of values obtained in each fraction (min to max) and the crossing lines indicate the mean. Four elongator tRNAs (top) or tRNA Met_i (bottom) were quantified in gradients prepared from 2 biological replicates of wild-type cells, 3 replicates of P TET-O7 -CDC48 and three replicates of dom34Δ P TET-O7 -CDC48 cells. ( C ) RNA in ribosomes pelleted from the 60S gradient fractions of the indicated strains was resolved on an acid-urea polyacrilamide gel and analyzed by northern hybridization using a radioactively labeled probe against tRNA Leu . Treatment with phenol (‘+’) was used to selectively remove long peptidyl-tRNAs. Prior to hybridization, the membrane was stained with methylene blue (MB) to control loading by visualizing 5S and 5.8S rRNAs in the 60S subunits. XC, the xylene cyanol band.

Techniques Used: Northern Blot, Hybridization, Labeling, Staining

37) Product Images from "Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae"

Article Title: Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091651

GFP-prApe1–labeled dots correspond to intact autophagosomes. (A) The 15,000× g pellet (P15) fraction from ypt7 Δ cells was treated with 1% Triton X-100 (TX), 0.5 mg/ml proteinase K (PK), or both for 30 minutes at 37°C before observation by fluorescence microscopy. Each fluorescence image of GFP-prApe1 was taken with the same exposure time. Bar represents 5 µm. (B) Detection of autophagosome by proteinase K-protection assay. Autophagosomes were collected into the P15 fraction (lanes 4–6). In ypt7 Δ atg1 Δ cells, all prApe1 was collected into the 15,000× g supernatant (S15) fraction (lanes 16–18). TX represents the presence of 1% Triton X-100, PK indicates the presence of 1 mg/ml proteinase K. prApe1 and dApe1 indicate precursor Ape1 and degraded Ape1, respectively. Mge1 is a mitochondrial matrix protein.
Figure Legend Snippet: GFP-prApe1–labeled dots correspond to intact autophagosomes. (A) The 15,000× g pellet (P15) fraction from ypt7 Δ cells was treated with 1% Triton X-100 (TX), 0.5 mg/ml proteinase K (PK), or both for 30 minutes at 37°C before observation by fluorescence microscopy. Each fluorescence image of GFP-prApe1 was taken with the same exposure time. Bar represents 5 µm. (B) Detection of autophagosome by proteinase K-protection assay. Autophagosomes were collected into the P15 fraction (lanes 4–6). In ypt7 Δ atg1 Δ cells, all prApe1 was collected into the 15,000× g supernatant (S15) fraction (lanes 16–18). TX represents the presence of 1% Triton X-100, PK indicates the presence of 1 mg/ml proteinase K. prApe1 and dApe1 indicate precursor Ape1 and degraded Ape1, respectively. Mge1 is a mitochondrial matrix protein.

Techniques Used: Labeling, Fluorescence, Microscopy

38) Product Images from "Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment"

Article Title: Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003355

Assembly of the p7 mutants is impaired prior to capsid envelopment. WT-Jc1- or mutant-transfected detergent-free cell lysates were subjected to a proteolytic digestion protection assay as follows. Lysates were separated into three aliquots which received different treatments: (i) left untreated, (ii) treated with 50 µg/ml proteinase K for 1 h on ice, or (iii) lysed in 5% Triton X-100 prior to proteinase K treatment (condition used for background correction). The amount of protease-resistant core was determined by Western Blot and ELISA. (A) Representative Western Blot stained for HCV core. (B) Western Blot signal intensities were quantified with LabImage 1D and values obtained for the proteinase K-treated sample were background-corrected and normalized to untreated control. Mean values and standard deviations of 3–6 independent experiments are shown.
Figure Legend Snippet: Assembly of the p7 mutants is impaired prior to capsid envelopment. WT-Jc1- or mutant-transfected detergent-free cell lysates were subjected to a proteolytic digestion protection assay as follows. Lysates were separated into three aliquots which received different treatments: (i) left untreated, (ii) treated with 50 µg/ml proteinase K for 1 h on ice, or (iii) lysed in 5% Triton X-100 prior to proteinase K treatment (condition used for background correction). The amount of protease-resistant core was determined by Western Blot and ELISA. (A) Representative Western Blot stained for HCV core. (B) Western Blot signal intensities were quantified with LabImage 1D and values obtained for the proteinase K-treated sample were background-corrected and normalized to untreated control. Mean values and standard deviations of 3–6 independent experiments are shown.

Techniques Used: Mutagenesis, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Characterization of differentially-sedimenting core complexes. Lysates of cells transfected with WT or mutant Jc1 were separated by rate zonal density gradient centrifugation. Each fraction was analyzed for infectivity by TCID 50  (A), core protein content by ELISA (B), and RNA copy number by qRT-PCR (C). Fractions 4 and 7 are highlighted with black arrows. (D) Fractions 4 and 7 were subjected to a proteolytic digestion protection assay and the amount of protease-resistant core protein was quantified by ELISA. The values obtained for the proteinase K-treated samples were background-subtracted and normalized to the untreated controls. Mean values and standard deviations of 2 independent experiments are shown. (E) Lysates of WT Jc1-transfected cells were separated by rate zonal density gradient centrifugation in the absence or presence of 1% DDM and core distribution along the gradient was determined by ELISA.
Figure Legend Snippet: Characterization of differentially-sedimenting core complexes. Lysates of cells transfected with WT or mutant Jc1 were separated by rate zonal density gradient centrifugation. Each fraction was analyzed for infectivity by TCID 50 (A), core protein content by ELISA (B), and RNA copy number by qRT-PCR (C). Fractions 4 and 7 are highlighted with black arrows. (D) Fractions 4 and 7 were subjected to a proteolytic digestion protection assay and the amount of protease-resistant core protein was quantified by ELISA. The values obtained for the proteinase K-treated samples were background-subtracted and normalized to the untreated controls. Mean values and standard deviations of 2 independent experiments are shown. (E) Lysates of WT Jc1-transfected cells were separated by rate zonal density gradient centrifugation in the absence or presence of 1% DDM and core distribution along the gradient was determined by ELISA.

Techniques Used: Transfection, Mutagenesis, Gradient Centrifugation, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Mutations in p7 affect capsid assembly. (A) WT-Jc1 or mutant-transfected cells were harvested 48 h post-transfection. Cell lysates were subjected to rate zonal density gradient centrifugation in the presence of non-ionic detergent (1% DDM) and core protein amounts along the gradient were measured by ELISA. Fractions 3 to 5 are highlighted with black arrows. The right panel shows the core amount relative to total core expression for fraction 5. (B) RNase digestion protection assay (see Materials and Methods ). Fractions 3 to 5 of the WT-Jc1 gradient were subjected to RNase digestion or left untreated. As a background control, samples were treated with proteinase K to remove protecting capsids, prior to RNase treatment. The amount of residual HCV RNA was quantified by qRT-PCR. In parallel crude HCV replication complexes (CRCs) were prepared by the method described by Quinkert et al. [62] . CRCs were then subjected to RNase treatment as described above. To mimic conditions of the sedimentation 1% DDM was added. (C) Separation of core complexes by blue-native-PAGE (4–16% gradient gel). (D) Two-dimensional gel electrophoresis of core complexes. Cell lysates were separated by blue native-PAGE (3–12% gradient gel) in a first electrophoresis and by SDS-PAGE in the second dimension. The results of three independent experiments are shown side by side. Gel pictures are shown vertically for easier comparison with the blue-native-PAGE results. (C, D) Core protein was detected by Western Blotting and subsequent immunodetection with the core-specific monoclonal antibody C7-50.
Figure Legend Snippet: Mutations in p7 affect capsid assembly. (A) WT-Jc1 or mutant-transfected cells were harvested 48 h post-transfection. Cell lysates were subjected to rate zonal density gradient centrifugation in the presence of non-ionic detergent (1% DDM) and core protein amounts along the gradient were measured by ELISA. Fractions 3 to 5 are highlighted with black arrows. The right panel shows the core amount relative to total core expression for fraction 5. (B) RNase digestion protection assay (see Materials and Methods ). Fractions 3 to 5 of the WT-Jc1 gradient were subjected to RNase digestion or left untreated. As a background control, samples were treated with proteinase K to remove protecting capsids, prior to RNase treatment. The amount of residual HCV RNA was quantified by qRT-PCR. In parallel crude HCV replication complexes (CRCs) were prepared by the method described by Quinkert et al. [62] . CRCs were then subjected to RNase treatment as described above. To mimic conditions of the sedimentation 1% DDM was added. (C) Separation of core complexes by blue-native-PAGE (4–16% gradient gel). (D) Two-dimensional gel electrophoresis of core complexes. Cell lysates were separated by blue native-PAGE (3–12% gradient gel) in a first electrophoresis and by SDS-PAGE in the second dimension. The results of three independent experiments are shown side by side. Gel pictures are shown vertically for easier comparison with the blue-native-PAGE results. (C, D) Core protein was detected by Western Blotting and subsequent immunodetection with the core-specific monoclonal antibody C7-50.

Techniques Used: Mutagenesis, Transfection, Gradient Centrifugation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Sedimentation, Blue Native PAGE, Two-Dimensional Gel Electrophoresis, Electrophoresis, SDS Page, Western Blot, Immunodetection

39) Product Images from "Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types"

Article Title: Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.090623

PrP Sc typing of sheep scrapie and human sporadic CJD. After treatment with proteinase K alone or combined with PNGase F, Western blot analysis of proteinase K digested classical scrapie cases ( A ) showed the typical triplet pattern of PrP Sc , whereas atypical/Nor98
Figure Legend Snippet: PrP Sc typing of sheep scrapie and human sporadic CJD. After treatment with proteinase K alone or combined with PNGase F, Western blot analysis of proteinase K digested classical scrapie cases ( A ) showed the typical triplet pattern of PrP Sc , whereas atypical/Nor98

Techniques Used: Western Blot

40) Product Images from "Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice"

Article Title: Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001257

Intranasal prion transmission is independent of lymphotoxin signaling. C57BL/6 mice treated with LTβR-Ig ( A ) or control muIgG ( B ), and mice lacking various components of the LT/TNF system ( D–F , as indicated) were intranasally inoculated with 4×10 5 LD 50 scrapie prions. Survival curves ( A, B, D, E and G ) and respective Western blots ( C, F and H ) indicate efficient prion infection and neuroinvasion. One animal that died early after intranasal inoculation (40 dpi) is reported as intercurrent death (i.d.) for reasons other than scrapie. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends used are as in Fig. 1H .
Figure Legend Snippet: Intranasal prion transmission is independent of lymphotoxin signaling. C57BL/6 mice treated with LTβR-Ig ( A ) or control muIgG ( B ), and mice lacking various components of the LT/TNF system ( D–F , as indicated) were intranasally inoculated with 4×10 5 LD 50 scrapie prions. Survival curves ( A, B, D, E and G ) and respective Western blots ( C, F and H ) indicate efficient prion infection and neuroinvasion. One animal that died early after intranasal inoculation (40 dpi) is reported as intercurrent death (i.d.) for reasons other than scrapie. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends used are as in Fig. 1H .

Techniques Used: Transmission Assay, Mouse Assay, Western Blot, Infection

Intranasal prion transmission in immnunodeficient mice. All mice were intranasally inoculated with 3×10 5 LD 50 prions. ( A ) C1q a −/− mice intranasally inoculated and ( B ) CD21 −/− mice intranasally inoculated are shown. Survival curves illustrate survival after intranasal prion challenge. Respective Western blots of C1qa −/− mice intranasally inoculated ( C, left panel ) and of CD21 −/− mice intranasally inoculated ( C, right panel ) are shown. Survival curves of CXCR5 −/− mice intranasally inoculated are shown ( D ). Respective Western blots of CXCR5 −/− mice intranasally inoculated. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends are as in Fig. 5 .
Figure Legend Snippet: Intranasal prion transmission in immnunodeficient mice. All mice were intranasally inoculated with 3×10 5 LD 50 prions. ( A ) C1q a −/− mice intranasally inoculated and ( B ) CD21 −/− mice intranasally inoculated are shown. Survival curves illustrate survival after intranasal prion challenge. Respective Western blots of C1qa −/− mice intranasally inoculated ( C, left panel ) and of CD21 −/− mice intranasally inoculated ( C, right panel ) are shown. Survival curves of CXCR5 −/− mice intranasally inoculated are shown ( D ). Respective Western blots of CXCR5 −/− mice intranasally inoculated. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends are as in Fig. 5 .

Techniques Used: Transmission Assay, Mouse Assay, Western Blot

Prion transmission by intranasal instillation. ( A ) Rag1 −/− mice intranasally inoculated with RML6 0.1%, ( B ) C57BL/6 mice that have been intranasally inoculated with 3×10 5 LD 50 prions. ( C ) Rag1 −/− mice i.c. inoculated with 3×10 5 LD 50 , ( D ) γ C Rag2 −/− mice intranasally inoculated with 4×10 5 LD 50 or ( E ) Balb/c mice intranasally inoculated with 4×10 5 LD 50 scrapie prions are shown. Survival curves ( A–D ) and respective Western blots ( F–G ) are indicative of efficient prion neuroinvasion. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Brain homogenates derived from a terminally scrapie-sick and a healthy C57BL/6 mouse served as positive and negative controls (s: sick; h: healthy), respectively. Molecular weights (kDa) are indicated on the left side of the blots. ( H and I ) Histopathological lesion severity score described as radar blot (astrogliosis, spongiform change and PrP Sc deposition) in 5 brain regions of both mouse lines exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter.
Figure Legend Snippet: Prion transmission by intranasal instillation. ( A ) Rag1 −/− mice intranasally inoculated with RML6 0.1%, ( B ) C57BL/6 mice that have been intranasally inoculated with 3×10 5 LD 50 prions. ( C ) Rag1 −/− mice i.c. inoculated with 3×10 5 LD 50 , ( D ) γ C Rag2 −/− mice intranasally inoculated with 4×10 5 LD 50 or ( E ) Balb/c mice intranasally inoculated with 4×10 5 LD 50 scrapie prions are shown. Survival curves ( A–D ) and respective Western blots ( F–G ) are indicative of efficient prion neuroinvasion. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Brain homogenates derived from a terminally scrapie-sick and a healthy C57BL/6 mouse served as positive and negative controls (s: sick; h: healthy), respectively. Molecular weights (kDa) are indicated on the left side of the blots. ( H and I ) Histopathological lesion severity score described as radar blot (astrogliosis, spongiform change and PrP Sc deposition) in 5 brain regions of both mouse lines exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter.

Techniques Used: Transmission Assay, Mouse Assay, Western Blot, Derivative Assay

PrP Sc deposition in brains of mice infected with prion aerosols and profiling of NSE-PrP mice. ( A ) Western blot analysis of brain homogenates (10%) from terminal or subclinical tg a 20 mice exposed to aerosols from 20% or 0.1% IBH for 10 min. PK+ or −: with or without proteinase K digest; kDa: Kilo Dalton. ( B–C ): Western blot analyses of brain homogenates from tg a 20 ( B ) or CD1 ( C ) mice exposed to prion aerosols from 20% IBH. ( D ) Histoblot analysis of brains from mice exposed to prion aerosols. Brains of tg a 20 mice challenged with aerosolized 10% (middle panel) or 20% (right panel) IBH showed deposits of PrP Sc in the cortex and mesencephalon. Because the brain of a Prnp o/o mouse showed no signal (left panel), we deduce that the signal in the middle and right panels represents local prion replication. ( E ) Histopathological lesion severity score analysis of 5 brain regions depicted as radar plots [51] (astrogliosis, spongiform change and PrP Sc deposition) derived from tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( F ) Histopathological lesion severity score of 5 brain regions shown as radar blot (astrogliosis, spongiform change and PrP Sc deposition) of i.c. prion inoculated tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice. (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( G ) Survival curve and ( H ) lesion severity scores of NSE-PrP mice exposed to a 20% aerosolized IBH for 10 min. ( I ) Histological and immunohistochemical characterization of scrapie-affected hippocampi of NSE-PrP mice after exposure to aerosolized 20% IBH. Stain legend as in Fig. 1H . Scale bar: 100µm.
Figure Legend Snippet: PrP Sc deposition in brains of mice infected with prion aerosols and profiling of NSE-PrP mice. ( A ) Western blot analysis of brain homogenates (10%) from terminal or subclinical tg a 20 mice exposed to aerosols from 20% or 0.1% IBH for 10 min. PK+ or −: with or without proteinase K digest; kDa: Kilo Dalton. ( B–C ): Western blot analyses of brain homogenates from tg a 20 ( B ) or CD1 ( C ) mice exposed to prion aerosols from 20% IBH. ( D ) Histoblot analysis of brains from mice exposed to prion aerosols. Brains of tg a 20 mice challenged with aerosolized 10% (middle panel) or 20% (right panel) IBH showed deposits of PrP Sc in the cortex and mesencephalon. Because the brain of a Prnp o/o mouse showed no signal (left panel), we deduce that the signal in the middle and right panels represents local prion replication. ( E ) Histopathological lesion severity score analysis of 5 brain regions depicted as radar plots [51] (astrogliosis, spongiform change and PrP Sc deposition) derived from tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( F ) Histopathological lesion severity score of 5 brain regions shown as radar blot (astrogliosis, spongiform change and PrP Sc deposition) of i.c. prion inoculated tg a 20 , CD1, C57BL/6 and 129SvxC57BL/6 mice. (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. ( G ) Survival curve and ( H ) lesion severity scores of NSE-PrP mice exposed to a 20% aerosolized IBH for 10 min. ( I ) Histological and immunohistochemical characterization of scrapie-affected hippocampi of NSE-PrP mice after exposure to aerosolized 20% IBH. Stain legend as in Fig. 1H . Scale bar: 100µm.

Techniques Used: Mouse Assay, Infection, Western Blot, Derivative Assay, Immunohistochemistry, Staining

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Negative Control:

Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella
Article Snippet: .. Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control. .. The samples were incubated at 37°C for 30 min, and protease activity was stopped by adding AEBSF [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] to each sample at final concentration of 0.5 mM.

Agarose Gel Electrophoresis:

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
Article Snippet: .. The reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science), and the DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate. .. The gel images were visualized using an FLA-7000 imaging analyzer (Fujifilm), and the band intensities were quantitated with the Multi Gauge software (Fujifilm).

Ethanol Precipitation:

Article Title: Alu RNP and Alu RNA regulate translation initiation in vitro
Article Snippet: .. Northern blotting RNA containing fractions were digested with proteinase K (Roche Diagnostics), 30 min at 55°C before ethanol precipitation in the presence of 5 µg glycogen. ..

Northern Blot:

Article Title: Alu RNP and Alu RNA regulate translation initiation in vitro
Article Snippet: .. Northern blotting RNA containing fractions were digested with proteinase K (Roche Diagnostics), 30 min at 55°C before ethanol precipitation in the presence of 5 µg glycogen. ..

Incubation:

Article Title: The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x
Article Snippet: .. The remaining beads were incubated with lysis buffer in the presence of (RNase-free) DNase (Roche) for 15 min at 37°C and washed three times with lysis buffer before incubation with 50 μg proteinase K (Roche) for an additional 15 min at 37°C. .. Coprecipitated RNA was then extracted by standard procedure and used for RT-PCR using BclX-1 and rtBclX-2 primers (Fig. S4).

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
Article Snippet: .. The reaction mixtures were further incubated at 37 °C for 10 min, and the reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science). .. The resulting DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate.

Article Title: Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies
Article Snippet: .. Briefly, 50 μl of membrane fractions resuspended in MELB with 4 μg ml−1 pepstatin A (Sigma) were pre-chilled to 0 °C then incubated with 30 μg ml−1 proteinase K (Roche) or trypsin (Sigma) on ice for 20 min, or 0.06 U μl−1 enterokinase (Merck Millipore) at room temperature for 2 h. The reactions were stopped by addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) and immunoblotted with antibodies to the Bak BH3 domain (4B5). ..

Imaging:

Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
Article Snippet: .. The reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science), and the DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate. .. The gel images were visualized using an FLA-7000 imaging analyzer (Fujifilm), and the band intensities were quantitated with the Multi Gauge software (Fujifilm).

Lysis:

Article Title: The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x
Article Snippet: .. The remaining beads were incubated with lysis buffer in the presence of (RNase-free) DNase (Roche) for 15 min at 37°C and washed three times with lysis buffer before incubation with 50 μg proteinase K (Roche) for an additional 15 min at 37°C. .. Coprecipitated RNA was then extracted by standard procedure and used for RT-PCR using BclX-1 and rtBclX-2 primers (Fig. S4).

Recombinant:

Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella
Article Snippet: .. Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control. .. The samples were incubated at 37°C for 30 min, and protease activity was stopped by adding AEBSF [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] to each sample at final concentration of 0.5 mM.

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    Roche proteinase k
    Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and <t>Proteinase</t> K prior to gel electrophoresis. Scale bar in B and C is 25 μm.
    Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: TF-ChIP Method for Tissue-Specific Gene Targets

    doi: 10.3389/fncel.2019.00095

    Figure Lengend Snippet: Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

    Article Snippet: Ethylenediaminetetraacetic acid (EDTA) (Sigma–Aldrich, United States; E5134) Tris (Roth, Germany; 5429.3) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma–Aldrich, United States; H3375) NaCl (Roth, Germany; 9265.2) Sodium deoxycholate (Sigma–Aldrich, United States; 30970) Triton X-100 (Sigma–Aldrich, United States; 93426) Tris–HCl (Roth, Germany; 9090) LiCl (Sigma–Aldrich, United States; L9650) Nonidet P-40 substitute (Sigma–Aldrich, United States; 74385) QubitTM dsDNA HS Assay Kit (Thermo Fisher, United States; Q32851) PierceTM Protein A/G Agarose (Thermo Fisher, United States; 20421) Antibodies: Rabbit anti-NICD (Cell Signaling, United States; #4147); Rabbit IgG (Cell Signaling, United States; #2729); and Rabbit anti-Acetyl-Histone H3 (Lys9) (Cell Signaling, United States; #9649) RNase A solution (Promega, United States; A7974) Proteinase K (Roche, Switzerland; 03508838103) Phenol/chloroform/isoamyl alcohol (Roth, Germany; A156) !Caution chloroform is toxic if absorbed through the skin, inhaled or ingested.

    Techniques: Expressing, Immunofluorescence, Transgenic Assay, Activation Assay, Staining, Sonication, Nucleic Acid Electrophoresis

    An HA ladder (lane 1), bSF (lane 2), and portions of blood/SF clots digested with proteinase K (lanes 3–7) were electrophoresed on a 1% agarose gel and stained with Stains-all.

    Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

    Article Title: The Biomechanical Properties of Mixtures of Blood and Synovial Fluid

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    Figure Lengend Snippet: An HA ladder (lane 1), bSF (lane 2), and portions of blood/SF clots digested with proteinase K (lanes 3–7) were electrophoresed on a 1% agarose gel and stained with Stains-all.

    Article Snippet: Clots of each sample mixture were prepared as above and digested with 0.5mg/mL proteinase K (Roche Applied Science, Indianapolis, IN) in PBE at 60°C overnight.

    Techniques: Agarose Gel Electrophoresis, Staining

    Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the  nirB  promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the  nirB  and  spiC  promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of  Salmonella  strain SL7207 pZ1204.

    Journal: Microbial Cell Factories

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

    doi: 10.1186/1475-2859-13-80

    Figure Lengend Snippet: Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the nirB promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the nirB and spiC promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of Salmonella strain SL7207 pZ1204.

    Article Snippet: Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control.

    Techniques: Expressing, Western Blot

    Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.

    Journal: Microbial Cell Factories

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

    doi: 10.1186/1475-2859-13-80

    Figure Lengend Snippet: Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.

    Article Snippet: Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control.

    Techniques: Western Blot, Expressing

    Endogenous Sam68 associates with the mRNAs encoding for regulators of apoptosis. 1 mg of HEK293 cell extracts was immunoprecipitated with 10 μg of either control rabbit IgGs or anti-Sam68 IgGs as described in Materials and methods. An aliquot of the immunoprecipitated proteins was analyzed in Western blot for the presence of Sam68 (A), and the remaining sample was extracted in phenol/chloroform after treatment with proteinase K and DNase. (B) Extracted RNA was retrotranscribed and used for PCR amplification with oligonucleotides specific for the indicated genes. (C) GST pull-down experiment using purified GST or GST-Sam68 immobilized on glutathione-agarose beads and 100 μg of total RNA extracted and purified from HEK293 cells. Adsorbed RNA was extracted, retrotranscribed, and amplified as described in B.

    Journal: The Journal of Cell Biology

    Article Title: The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

    doi: 10.1083/jcb.200701005

    Figure Lengend Snippet: Endogenous Sam68 associates with the mRNAs encoding for regulators of apoptosis. 1 mg of HEK293 cell extracts was immunoprecipitated with 10 μg of either control rabbit IgGs or anti-Sam68 IgGs as described in Materials and methods. An aliquot of the immunoprecipitated proteins was analyzed in Western blot for the presence of Sam68 (A), and the remaining sample was extracted in phenol/chloroform after treatment with proteinase K and DNase. (B) Extracted RNA was retrotranscribed and used for PCR amplification with oligonucleotides specific for the indicated genes. (C) GST pull-down experiment using purified GST or GST-Sam68 immobilized on glutathione-agarose beads and 100 μg of total RNA extracted and purified from HEK293 cells. Adsorbed RNA was extracted, retrotranscribed, and amplified as described in B.

    Article Snippet: The remaining beads were incubated with lysis buffer in the presence of (RNase-free) DNase (Roche) for 15 min at 37°C and washed three times with lysis buffer before incubation with 50 μg proteinase K (Roche) for an additional 15 min at 37°C.

    Techniques: Immunoprecipitation, Western Blot, Polymerase Chain Reaction, Amplification, Purification