Structured Review

Carl Roth GmbH proteinase k
Sgk1 associates with the OMM ( A ) Membrane association of imported radiolabelled Sgk1 was assessed by preparation of mitochondrial (mitos) soluble (s/n) and membrane (mem) fractions. Radiolabelled proteins were visualized by autoradiography (AR). The peripheral membrane protein cytochrome c (CytC) was released by Na 2 CO 3 treatment and visualized by Western blot (WB, lower panel). Star symbol: this signal sometimes occurred after in vitro translation and is most likely a degradation product (also in B ). ( B ) After import of in vitro -translated radiolabelled Sgk1, mitochondria were treated with 200 μg/ml <t>proteinase</t> K (PK) in the presence or absence of Triton X-100 or left untreated. Sgk1 was subject to complete digestion, indicating a localization to the cytoplasmic face of the OMM. ivt; an aliquot of the in vitro translation product. ( C ) Aliquots of the protease protection assay were subjected to Western-blot analyses with antibodies directed against the matrix protein Hsp60, and the intermembrane space proteins Smac/DIABLO and cytochrome c ], and therefore served as loading control. Hsp60 and Smac/DIABLO remain intact in the absence of Triton X-100, demonstrating the integrity of the organelles. ( D ) The capability of the preparation to import proteins correctly is shown by the import of in vitro -translated fumarate hydratase (FH) in parallel. Imported fumarate hydratase migrates slightly faster than the in vitro translation product (ivt) because of the removal of the signal peptide by the processing peptidase after import into the matrix.
Proteinase K, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "The N-terminus of the serum- and glucocorticoid-inducible kinase Sgk1 specifies mitochondrial localization and rapid turnover"

Article Title: The N-terminus of the serum- and glucocorticoid-inducible kinase Sgk1 specifies mitochondrial localization and rapid turnover

Journal: Biochemical Journal

doi: 10.1042/BJ20060386

Sgk1 associates with the OMM ( A ) Membrane association of imported radiolabelled Sgk1 was assessed by preparation of mitochondrial (mitos) soluble (s/n) and membrane (mem) fractions. Radiolabelled proteins were visualized by autoradiography (AR). The peripheral membrane protein cytochrome c (CytC) was released by Na 2 CO 3 treatment and visualized by Western blot (WB, lower panel). Star symbol: this signal sometimes occurred after in vitro translation and is most likely a degradation product (also in B ). ( B ) After import of in vitro -translated radiolabelled Sgk1, mitochondria were treated with 200 μg/ml proteinase K (PK) in the presence or absence of Triton X-100 or left untreated. Sgk1 was subject to complete digestion, indicating a localization to the cytoplasmic face of the OMM. ivt; an aliquot of the in vitro translation product. ( C ) Aliquots of the protease protection assay were subjected to Western-blot analyses with antibodies directed against the matrix protein Hsp60, and the intermembrane space proteins Smac/DIABLO and cytochrome c ], and therefore served as loading control. Hsp60 and Smac/DIABLO remain intact in the absence of Triton X-100, demonstrating the integrity of the organelles. ( D ) The capability of the preparation to import proteins correctly is shown by the import of in vitro -translated fumarate hydratase (FH) in parallel. Imported fumarate hydratase migrates slightly faster than the in vitro translation product (ivt) because of the removal of the signal peptide by the processing peptidase after import into the matrix.
Figure Legend Snippet: Sgk1 associates with the OMM ( A ) Membrane association of imported radiolabelled Sgk1 was assessed by preparation of mitochondrial (mitos) soluble (s/n) and membrane (mem) fractions. Radiolabelled proteins were visualized by autoradiography (AR). The peripheral membrane protein cytochrome c (CytC) was released by Na 2 CO 3 treatment and visualized by Western blot (WB, lower panel). Star symbol: this signal sometimes occurred after in vitro translation and is most likely a degradation product (also in B ). ( B ) After import of in vitro -translated radiolabelled Sgk1, mitochondria were treated with 200 μg/ml proteinase K (PK) in the presence or absence of Triton X-100 or left untreated. Sgk1 was subject to complete digestion, indicating a localization to the cytoplasmic face of the OMM. ivt; an aliquot of the in vitro translation product. ( C ) Aliquots of the protease protection assay were subjected to Western-blot analyses with antibodies directed against the matrix protein Hsp60, and the intermembrane space proteins Smac/DIABLO and cytochrome c ], and therefore served as loading control. Hsp60 and Smac/DIABLO remain intact in the absence of Triton X-100, demonstrating the integrity of the organelles. ( D ) The capability of the preparation to import proteins correctly is shown by the import of in vitro -translated fumarate hydratase (FH) in parallel. Imported fumarate hydratase migrates slightly faster than the in vitro translation product (ivt) because of the removal of the signal peptide by the processing peptidase after import into the matrix.

Techniques Used: Autoradiography, Western Blot, In Vitro

2) Product Images from "CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs"

Article Title: CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

Journal: Scientific Reports

doi: 10.1038/srep31313

Evidence for distinct circRNA-protein complexes in mammalian cells. ( A ) Sedimentation profiles of circRNPs/circRNAs. Cytoplasmic S100 extract and corresponding free RNA from HeLa cells were fractionated by glycerol gradient centrifugation (#1–22 from top to bottom; the last fraction containing the resuspended pellet), followed by RT-PCR analysis of 12 abundant circRNAs across the gradient (ordered from top to bottom according to their sizes; given in nucleotides). The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). ( B ) Quantitation of circRNA distribution across gradient, comparing free RNA prepared from extract (RNA, in blue), cytoplasmic S100 fraction (extract, in red), and proteinase K-treated extract (extract + PK, in dashed lines). Data from panel A (RNA/extract) and from  Supplementary Fig. S1A  (extract + PK) were used for quantitation.
Figure Legend Snippet: Evidence for distinct circRNA-protein complexes in mammalian cells. ( A ) Sedimentation profiles of circRNPs/circRNAs. Cytoplasmic S100 extract and corresponding free RNA from HeLa cells were fractionated by glycerol gradient centrifugation (#1–22 from top to bottom; the last fraction containing the resuspended pellet), followed by RT-PCR analysis of 12 abundant circRNAs across the gradient (ordered from top to bottom according to their sizes; given in nucleotides). The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). ( B ) Quantitation of circRNA distribution across gradient, comparing free RNA prepared from extract (RNA, in blue), cytoplasmic S100 fraction (extract, in red), and proteinase K-treated extract (extract + PK, in dashed lines). Data from panel A (RNA/extract) and from Supplementary Fig. S1A (extract + PK) were used for quantitation.

Techniques Used: Sedimentation, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

IMP3 protein specifically and stably bound to circRNA family: validation and characterization. ( A ) Validation of IMP3 bound to specific circRNAs. Lysates of HepG2, PANC1, and PATU cells were subjected to immunoprecipitation with anti-IMP3 antibodies (IP), or as mock control, with anti-FLAG antibodies (m). Co-precipitated RNA was purified and assayed by RT-PCR for FTL mRNA (positive control), CAMSAP1 (negative control circRNA), and for the putative IMP3-associated circRNAs CDYL, NFATC3, and ANKRD17. For each of the circRNAs, the linear isoform of the respective gene was tested in addition (circ/lin; 90% of the mock- and IMP3-immunoprecipitates were used in RT-PCR). In addition, 5% of the input material was assayed (I). M, markers (in bp). ( B ) Quantitative immunoprecipitation analysis of IMP3-circRNA association in three cell lines (HepG2, PANC1, and PATU). For the same set of IMP3 circRNA targets and controls as shown in panel A, the immunoprecipitation efficiences were determined by RT-qPCR assays (% of input; statistical deviations based on biological duplicates). ( C ) Sedimentation profiles of IMP3-containing circRNPs. Cytoplasmic S100 extract from HeLa cells (extract), corresponding free RNA (RNA), and proteinase K-treated extract (extract + PK) were fractionated by glycerol gradient centrifugation (#1–22; the last fraction contains the resuspended pellet), followed by RT-PCR analysis of two IMP3-containing circRNAs (NFATC3 and ANKRD17). The relatively low recovery of circRNAs NFATC3 (1298 nt) and especially ANKRD17 (1832 nt), the largest circRNAs analyzed in S100 extract, may be caused by the higher tendency of such large circRNPs to aggregate and form precipitates, which were lost in the pellet fraction. The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). For comparison, the distribution of total IMP3 protein across the gradient was visualized by Western blotting in extract and, as a control, in proteinase K-treated extract. ( D ) IMP3 immunoprecipitation efficiencies of gradient-purified NFATC3 and ANKRD17 circRNPs. HeLa cytoplasmic S100 extract was gradient-fractionated, and NFATC3 and ANKRD17 circRNPs were IMP3-immunoprecipitated from the respective peak fractions (NFATC3, #15; ANKRD17, #17), using CAMSAP1 circRNA (peak fraction #11) and anti-eIF4E immunoprecipitation as negative controls. Immunoprecipitation efficiencies (% of input; statistical deviations based on technical triplicates) were determined by RT-qPCR.
Figure Legend Snippet: IMP3 protein specifically and stably bound to circRNA family: validation and characterization. ( A ) Validation of IMP3 bound to specific circRNAs. Lysates of HepG2, PANC1, and PATU cells were subjected to immunoprecipitation with anti-IMP3 antibodies (IP), or as mock control, with anti-FLAG antibodies (m). Co-precipitated RNA was purified and assayed by RT-PCR for FTL mRNA (positive control), CAMSAP1 (negative control circRNA), and for the putative IMP3-associated circRNAs CDYL, NFATC3, and ANKRD17. For each of the circRNAs, the linear isoform of the respective gene was tested in addition (circ/lin; 90% of the mock- and IMP3-immunoprecipitates were used in RT-PCR). In addition, 5% of the input material was assayed (I). M, markers (in bp). ( B ) Quantitative immunoprecipitation analysis of IMP3-circRNA association in three cell lines (HepG2, PANC1, and PATU). For the same set of IMP3 circRNA targets and controls as shown in panel A, the immunoprecipitation efficiences were determined by RT-qPCR assays (% of input; statistical deviations based on biological duplicates). ( C ) Sedimentation profiles of IMP3-containing circRNPs. Cytoplasmic S100 extract from HeLa cells (extract), corresponding free RNA (RNA), and proteinase K-treated extract (extract + PK) were fractionated by glycerol gradient centrifugation (#1–22; the last fraction contains the resuspended pellet), followed by RT-PCR analysis of two IMP3-containing circRNAs (NFATC3 and ANKRD17). The relatively low recovery of circRNAs NFATC3 (1298 nt) and especially ANKRD17 (1832 nt), the largest circRNAs analyzed in S100 extract, may be caused by the higher tendency of such large circRNPs to aggregate and form precipitates, which were lost in the pellet fraction. The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). For comparison, the distribution of total IMP3 protein across the gradient was visualized by Western blotting in extract and, as a control, in proteinase K-treated extract. ( D ) IMP3 immunoprecipitation efficiencies of gradient-purified NFATC3 and ANKRD17 circRNPs. HeLa cytoplasmic S100 extract was gradient-fractionated, and NFATC3 and ANKRD17 circRNPs were IMP3-immunoprecipitated from the respective peak fractions (NFATC3, #15; ANKRD17, #17), using CAMSAP1 circRNA (peak fraction #11) and anti-eIF4E immunoprecipitation as negative controls. Immunoprecipitation efficiencies (% of input; statistical deviations based on technical triplicates) were determined by RT-qPCR.

Techniques Used: Stable Transfection, Immunoprecipitation, Purification, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Quantitative RT-PCR, Sedimentation, Gradient Centrifugation, Western Blot

3) Product Images from "Developmental differences in genome replication program and origin activation"

Article Title: Developmental differences in genome replication program and origin activation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkaa1124

DNA replication fiber and genome size analysis in mouse embryonic stem cells. ( A ) Schematic outline of the experimental setup for DNA fiber analysis. mES cells were sequentially labeled with IdU and CldU for 15 min, harvested and embedded in agarose. After a proteinase K digestion step, agarose was digested and high molecular weight naked DNA was stretched on silanized glass coverslips. Nucleotide analogs and single stranded DNA (ssDNA) were immunofluorescently detected. ( B–E ) The length of fluorescent tracks of the second pulse (CldU) were measured (1 μm ∼ 2000 nucleotides, Supplementary Figure S6 ) and the mean replication fork speed (RFS, ( B )), inter-origin distance (IOD, ( C )), percentage of unidirectional forks ( F ) and asymmetry of bidirectional forks ( G ) were calculated as indicated in (A). Additionally, the percentages of forks within a given range of RFS are indicated in (B). For comparison of the RFS of the ‘left’ and ‘right’ fork of a bidirectional fork, RFS values were plotted in a scatterplot. The solid grey line represents the linear relation x = y and dotted lines represent thresholds allowing for a 35% (± StDev calculated for the asymmetry factor) difference between lengths of the two forks. ( D ) Visual representation of origin mapping in two arbitrarily selected regions (mouse (Mus musculus, mm10) chromosomes 9 and 11). SNS-seq origin profiles and identified origins in mES and MEF cells are shown. OK-seq origin profiles in activated (act.) B cells, called peaks along with the middle point of each peak are represented. Replication profile scale is indicated in the upper left corner. The comparison between identified and clustered origin peaks is shown in Supplementary Figure S20 . ( E ) IOD distributions based on the genome-wide origin maps in mES cells and mouse embryonic fibroblast (MEF) are shown (SNS-seq). The sequencing datasets used for the analysis include two independent replicates of origin mapping for each condition. ( H ) Ploidy of J1 mES cells was determined via karyotype analysis of metaphase spreads. For genome size calculation, the sizes of individual mouse chromosomes (19 autosomes + X and Y chromosomes) were retrieved from the Genome Reference Consortium database. Additionally, genome sizes measured by flow cytometry are indicated. Boxplots/violinplots are as in Figure 2 and Supplementary Figure S7 . Statistical details are depicted in the plots or summarized in Supplementary Table S13 . All experiments were done in at least two independent biological replicates. Black dots within box/violin plots represent mean values. * P
Figure Legend Snippet: DNA replication fiber and genome size analysis in mouse embryonic stem cells. ( A ) Schematic outline of the experimental setup for DNA fiber analysis. mES cells were sequentially labeled with IdU and CldU for 15 min, harvested and embedded in agarose. After a proteinase K digestion step, agarose was digested and high molecular weight naked DNA was stretched on silanized glass coverslips. Nucleotide analogs and single stranded DNA (ssDNA) were immunofluorescently detected. ( B–E ) The length of fluorescent tracks of the second pulse (CldU) were measured (1 μm ∼ 2000 nucleotides, Supplementary Figure S6 ) and the mean replication fork speed (RFS, ( B )), inter-origin distance (IOD, ( C )), percentage of unidirectional forks ( F ) and asymmetry of bidirectional forks ( G ) were calculated as indicated in (A). Additionally, the percentages of forks within a given range of RFS are indicated in (B). For comparison of the RFS of the ‘left’ and ‘right’ fork of a bidirectional fork, RFS values were plotted in a scatterplot. The solid grey line represents the linear relation x = y and dotted lines represent thresholds allowing for a 35% (± StDev calculated for the asymmetry factor) difference between lengths of the two forks. ( D ) Visual representation of origin mapping in two arbitrarily selected regions (mouse (Mus musculus, mm10) chromosomes 9 and 11). SNS-seq origin profiles and identified origins in mES and MEF cells are shown. OK-seq origin profiles in activated (act.) B cells, called peaks along with the middle point of each peak are represented. Replication profile scale is indicated in the upper left corner. The comparison between identified and clustered origin peaks is shown in Supplementary Figure S20 . ( E ) IOD distributions based on the genome-wide origin maps in mES cells and mouse embryonic fibroblast (MEF) are shown (SNS-seq). The sequencing datasets used for the analysis include two independent replicates of origin mapping for each condition. ( H ) Ploidy of J1 mES cells was determined via karyotype analysis of metaphase spreads. For genome size calculation, the sizes of individual mouse chromosomes (19 autosomes + X and Y chromosomes) were retrieved from the Genome Reference Consortium database. Additionally, genome sizes measured by flow cytometry are indicated. Boxplots/violinplots are as in Figure 2 and Supplementary Figure S7 . Statistical details are depicted in the plots or summarized in Supplementary Table S13 . All experiments were done in at least two independent biological replicates. Black dots within box/violin plots represent mean values. * P

Techniques Used: Labeling, Molecular Weight, Genome Wide, Sequencing, Flow Cytometry

4) Product Images from "Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation"

Article Title: Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004365

The inhibition of TNF-induced IP-10 secretion is mediated by a surface protein of L. casei. (A) Mode-K cells were stimulated for 24 h with L. casei , Lactobacillus plantarum 299v or E. coli Nissle 1917 (moi as indicated) with or without TNF (10 ng/ml) activation for 24 h. The concentration of IP-10 and IL-6 in cell culture supernatants was measured by ELISA. Bars represent mean values (+/− SD) of triplicate samples. (B) Mode-K cells were stimulated with L. casei (moi 20) killed by formaldehyde fixation (fix), lysozyme (lys)-digestion or heat (heat)-treatment with or without TNF (10 ng/ml) activation for 24 h. (C) L. casei was treated with trypsin (try), proteinase K (pK) or phospholipase A (pA) followed by formaldehyde-fixation (fL.c). (D) TLR2 deficient as well as TLR2 expressing HEK293 cells (absence/presence of TLR2 in the cells was analysed by real-time PCR) were stimulated for 24 h with TNF (10 ng/ml) and L. casei (moi 20). IP-10 concentration was measured in the culture supernatants by ELISA. The bars represent mean values (+/− SD) of triplicate samples.
Figure Legend Snippet: The inhibition of TNF-induced IP-10 secretion is mediated by a surface protein of L. casei. (A) Mode-K cells were stimulated for 24 h with L. casei , Lactobacillus plantarum 299v or E. coli Nissle 1917 (moi as indicated) with or without TNF (10 ng/ml) activation for 24 h. The concentration of IP-10 and IL-6 in cell culture supernatants was measured by ELISA. Bars represent mean values (+/− SD) of triplicate samples. (B) Mode-K cells were stimulated with L. casei (moi 20) killed by formaldehyde fixation (fix), lysozyme (lys)-digestion or heat (heat)-treatment with or without TNF (10 ng/ml) activation for 24 h. (C) L. casei was treated with trypsin (try), proteinase K (pK) or phospholipase A (pA) followed by formaldehyde-fixation (fL.c). (D) TLR2 deficient as well as TLR2 expressing HEK293 cells (absence/presence of TLR2 in the cells was analysed by real-time PCR) were stimulated for 24 h with TNF (10 ng/ml) and L. casei (moi 20). IP-10 concentration was measured in the culture supernatants by ELISA. The bars represent mean values (+/− SD) of triplicate samples.

Techniques Used: Inhibition, Activation Assay, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

Related Articles

Centrifugation:

Article Title: Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts
Article Snippet: Supernatants were freeze-dried and dissolved in 300 µL H2 Odd , substituted with 900 µL ethanol (96%, denatured) and incubated overnight at −20 °C. .. After centrifugation (5000 rpm, 5 min, 4 °C), supernatants were discarded and the pellet was dissolved in 300 µL 100 mM ammoniumacetat (T872, Carl-Roth, Karlsruhe, Germany) supplemented with 0.107 U proteinase K (P4850, Sigma-Aldrich, Steinheim, Germany) and incubated for 2 h on a rotary shaker (55 °C, 700 rpm), followed by proteinase K inactivation at 100 °C for 5 min. After the substitution of 900 µL ethanol (96%, denatured), samples were stored overnight at −20 °C and centrifuged (5000 rpm, 5 min, 4 °C) the next day. ..

Incubation:

Article Title: Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts
Article Snippet: Supernatants were freeze-dried and dissolved in 300 µL H2 Odd , substituted with 900 µL ethanol (96%, denatured) and incubated overnight at −20 °C. .. After centrifugation (5000 rpm, 5 min, 4 °C), supernatants were discarded and the pellet was dissolved in 300 µL 100 mM ammoniumacetat (T872, Carl-Roth, Karlsruhe, Germany) supplemented with 0.107 U proteinase K (P4850, Sigma-Aldrich, Steinheim, Germany) and incubated for 2 h on a rotary shaker (55 °C, 700 rpm), followed by proteinase K inactivation at 100 °C for 5 min. After the substitution of 900 µL ethanol (96%, denatured), samples were stored overnight at −20 °C and centrifuged (5000 rpm, 5 min, 4 °C) the next day. ..

Article Title: Saccharomyces cerevisiae
Article Snippet: Protein concentrations were determined with the colorimetric DC protein assay (Bio-Rad) using bovine serum albumin as the standard. .. 25 μg of fresh enzymatically isolated mt proteins were incubated with 2 μg/ml Proteinase K solution (Invitrogen) in buffer (650 m m sorbitol, 10 m m Tris-HCl, pH 7.4) in the presence or absence of 1% Triton X-100 (Roth) for 15 min on ice. .. Proteinase K activity was stopped by adding AEBSF (1 m m ) and PI-Mix (1×).

Isolation:

Article Title: Saccharomyces cerevisiae
Article Snippet: Protein concentrations were determined with the colorimetric DC protein assay (Bio-Rad) using bovine serum albumin as the standard. .. 25 μg of fresh enzymatically isolated mt proteins were incubated with 2 μg/ml Proteinase K solution (Invitrogen) in buffer (650 m m sorbitol, 10 m m Tris-HCl, pH 7.4) in the presence or absence of 1% Triton X-100 (Roth) for 15 min on ice. .. Proteinase K activity was stopped by adding AEBSF (1 m m ) and PI-Mix (1×).

other:

Article Title: Effects of Lysozyme, Proteinase K, and Cephalosporins on Biofilm Formation by Clinical Isolates of Pseudomonas aeruginosa
Article Snippet: Effects of Lysozyme and Proteinase K on Biofilm Formation The effects of lysozyme (Carl Roth, Karlsruhe, Germany) and proteinase K (QIAGEN, cat. no. 19131) on the biofilm formation ability of the nine most strongly adherent P . aeruginosa isolates (highest values) were investigated by the biofilm quantitative assay, as mentioned in the previous section.

Lysis:

Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
Article Snippet: .. Lysis of the tissue was performed by adding the same volume of a solution composed of 2 mg/ml proteinase K (Carl Roth, 7528.3), 0.5% sodium dodecyl sulfate (Carl Roth, CN30) and 10 mM EDTA, and incubating at 37°C for 2 h. The genomic DNA was precipitated with isopropanol, dissolved in H2 0, and subjected to quantitative RT-PCR with mouse-specific primers as listed below. .. HEK 293 cells were cultured in DMEM/F-12 (Thermo Fisher Scientific, 31331-028) with 10% fetal calf serum (FCS; Thermo Fisher Scientific, 10270-106) and 1% penicillin/streptomycin (Thermo Fisher Scientific, 15140-163).

Quantitative RT-PCR:

Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
Article Snippet: .. Lysis of the tissue was performed by adding the same volume of a solution composed of 2 mg/ml proteinase K (Carl Roth, 7528.3), 0.5% sodium dodecyl sulfate (Carl Roth, CN30) and 10 mM EDTA, and incubating at 37°C for 2 h. The genomic DNA was precipitated with isopropanol, dissolved in H2 0, and subjected to quantitative RT-PCR with mouse-specific primers as listed below. .. HEK 293 cells were cultured in DMEM/F-12 (Thermo Fisher Scientific, 31331-028) with 10% fetal calf serum (FCS; Thermo Fisher Scientific, 10270-106) and 1% penicillin/streptomycin (Thermo Fisher Scientific, 15140-163).

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    Carl Roth GmbH proteinase k
    Western blot analysis of short-time incubation experiments. a) Western blot detection of PrP C extracted from soil mixed with non-infectious brain homogenate (5% pork brain in German standard soil). Several different buffers and solutions were used for extraction. Lane 1: water; lane 2: Triton X-100; lane 3: 1% urea; lane 4: 1% SDS; lane 5: Zwittergent; lane 6: RIPA buffer; lane 7: NP-40; lane 8: Na-sarcosyl. b) Western blot detection of PrP27-30, the proteinase K-resistant core of PrP Sc , extracted by using 1% SDS from soil contaminated with 263 K scrapie brain homogenate from hamsters after 1 h of incubation (dilution series). PrP Sc could be detected in soil samples containing 1.25 µg or higher amounts of scrapie brain tissue after extraction with 1% SDS-solution. Samples were digested with <t>proteinase</t> K prior to Western blotting.
    Proteinase K, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k/product/Carl Roth GmbH
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2021-03
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    Western blot analysis of short-time incubation experiments. a) Western blot detection of PrP C extracted from soil mixed with non-infectious brain homogenate (5% pork brain in German standard soil). Several different buffers and solutions were used for extraction. Lane 1: water; lane 2: Triton X-100; lane 3: 1% urea; lane 4: 1% SDS; lane 5: Zwittergent; lane 6: RIPA buffer; lane 7: NP-40; lane 8: Na-sarcosyl. b) Western blot detection of PrP27-30, the proteinase K-resistant core of PrP Sc , extracted by using 1% SDS from soil contaminated with 263 K scrapie brain homogenate from hamsters after 1 h of incubation (dilution series). PrP Sc could be detected in soil samples containing 1.25 µg or higher amounts of scrapie brain tissue after extraction with 1% SDS-solution. Samples were digested with proteinase K prior to Western blotting.

    Journal: PLoS ONE

    Article Title: Scrapie Agent (Strain 263K) Can Transmit Disease via the Oral Route after Persistence in Soil over Years

    doi: 10.1371/journal.pone.0000435

    Figure Lengend Snippet: Western blot analysis of short-time incubation experiments. a) Western blot detection of PrP C extracted from soil mixed with non-infectious brain homogenate (5% pork brain in German standard soil). Several different buffers and solutions were used for extraction. Lane 1: water; lane 2: Triton X-100; lane 3: 1% urea; lane 4: 1% SDS; lane 5: Zwittergent; lane 6: RIPA buffer; lane 7: NP-40; lane 8: Na-sarcosyl. b) Western blot detection of PrP27-30, the proteinase K-resistant core of PrP Sc , extracted by using 1% SDS from soil contaminated with 263 K scrapie brain homogenate from hamsters after 1 h of incubation (dilution series). PrP Sc could be detected in soil samples containing 1.25 µg or higher amounts of scrapie brain tissue after extraction with 1% SDS-solution. Samples were digested with proteinase K prior to Western blotting.

    Article Snippet: 1 h, followed by a centrifugation step at 5,000 rpm for 20 min. 200 µl of the clear supernatant was incubated with proteinase K (50 µg/ml; 37°C; 1 h, Carl Roth GmbH, Karlsruhe, Germany) to eliminate non-resistant proteins.

    Techniques: Western Blot, Incubation

    Integrity of the mitochondrial inner membrane. a Mitochondrial protein steady state levels were analyzed by SDS-PAGE and western blotting with antibodies directed against the indicated proteins. b Isolated mitochondria suspended in potassium chloride based isolation buffer were treated with Proteinase K (Prot. K) and analyzed as described in panel ( a )

    Journal: Metabolomics

    Article Title: Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

    doi: 10.1007/s11306-018-1352-x

    Figure Lengend Snippet: Integrity of the mitochondrial inner membrane. a Mitochondrial protein steady state levels were analyzed by SDS-PAGE and western blotting with antibodies directed against the indicated proteins. b Isolated mitochondria suspended in potassium chloride based isolation buffer were treated with Proteinase K (Prot. K) and analyzed as described in panel ( a )

    Article Snippet: Proteinase K was inactivated by addition of 2.4 mM PMSF (Phenylmethylsulfonylfluorid, Roth) and further incubated on ice for 10 min. Mitochondria were pelleted for 10 min at 20,817×g (14,000 rpm, 5417R, Eppendorf) at 4 °C and washed with 200 µL isolation buffer containing 1 mM PMSF.

    Techniques: SDS Page, Western Blot, Isolation

    CSNK2 phosphorylates TOMM22 and the absence of Csnk2b compromises CSNK2 catalytical activity and protein amount, but neither is required for TOMM complex biogenesis nor mitochondrial protein import. (A) Mouse TOMM22 was purified and used for in vitro radio-isotope-assisted phosphorylation with different kinases. The image of the autoradiogram shows that mouse TOMM22 was only phosphorylated in the presence of protein kinase CSNK2 and is not phosphorylated by any of the other kinases used. The amount of histidine-tagged TOMM22 served as loading control. (B) In vitro radio-isotope-assisted phosphorylation of TOMM22 is less efficient by muscle lysates of csnk2b ∆/∆ (D) In vitro phosphorylation experiments were performed with purified mouse TOMM22 wild-type protein and its alanine mutants together with recombinant CSNK2 using radiolabeled ATP. (E) T7 tagged TOMM22 wild-type and alanine-mutant expression plasmids were transfected into cultured cells, protein lysates immunprecipitated by a T7-specific antibody, precipitates were resolved by SDS-PAGE, and western blot membranes incubated with either a T7 or a TOMM22-p-S15-specific antibody. Note, the TOMM22-p-S15-specific antibody detects wild-type TOMM22, but not the TOMM22 S15A or TOMM22 S15A,T43A mutants. (F) The amount of catalytic activity-containing CSNK2A1 and CSNK2A2 subunits was determined in the absence of Csnk2b in skeletal muscle fiber lysates. Skeletal muscles soleus and tibialis anterior were used from approximately 2- (n = 3 mice per genotype) and 6- to 8-mo-old (n = 3 mice per genotype) mice. Obviously, CSNK2B protein is absent in csnk2b ∆/∆ , HSA-Cre muscle lysates. ACTN2 served as loading control. (G and H) Graphs represent protein amounts of CSNK2 subunits which were analyzed before by western blot (F). Note, in response to the absence of Csnk2b [ 35 S]-radiolabeled yeast proteins Cox4, Mdh1 and Atp2 were individually imported into mitochondria (Δψ, membrane potential). Mitochondria were treated with proteinase K and analyzed by SDS-PAGE. p, precursor; m, mature. Import into mitochondria after the longest import time was set to 100% (control). Note, all mitochondrial precursor (p) proteins are imported into mitochondria and processed to shorter mature (m) size with similar time kinetic between control and the csnk2b ∆/∆ , HSA-Cre indicating mitochondrial protein import in vitro being unaffected. (J) Mitochondria were isolated from skeletal muscles of adult wild-type or csnk2b ∆/∆ , HSA-Cre mice and equal amounts of native TOMM complexes were resolved by blue-native gels (BN) which are used to separate native protein complexes. After western blot, different TOMM family members within native TOMM complexes were detected by specific antibodies as shown by representative images. The amount of these TOMM family members is similar between TOMM complexes from mitochondria of wild-type or csnk2b ∆/∆ , HSA-Cre muscles, indicating proper biogenesis of TOMM complexes.

    Journal: Autophagy

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy

    doi: 10.1080/15548627.2017.1403716

    Figure Lengend Snippet: CSNK2 phosphorylates TOMM22 and the absence of Csnk2b compromises CSNK2 catalytical activity and protein amount, but neither is required for TOMM complex biogenesis nor mitochondrial protein import. (A) Mouse TOMM22 was purified and used for in vitro radio-isotope-assisted phosphorylation with different kinases. The image of the autoradiogram shows that mouse TOMM22 was only phosphorylated in the presence of protein kinase CSNK2 and is not phosphorylated by any of the other kinases used. The amount of histidine-tagged TOMM22 served as loading control. (B) In vitro radio-isotope-assisted phosphorylation of TOMM22 is less efficient by muscle lysates of csnk2b ∆/∆ (D) In vitro phosphorylation experiments were performed with purified mouse TOMM22 wild-type protein and its alanine mutants together with recombinant CSNK2 using radiolabeled ATP. (E) T7 tagged TOMM22 wild-type and alanine-mutant expression plasmids were transfected into cultured cells, protein lysates immunprecipitated by a T7-specific antibody, precipitates were resolved by SDS-PAGE, and western blot membranes incubated with either a T7 or a TOMM22-p-S15-specific antibody. Note, the TOMM22-p-S15-specific antibody detects wild-type TOMM22, but not the TOMM22 S15A or TOMM22 S15A,T43A mutants. (F) The amount of catalytic activity-containing CSNK2A1 and CSNK2A2 subunits was determined in the absence of Csnk2b in skeletal muscle fiber lysates. Skeletal muscles soleus and tibialis anterior were used from approximately 2- (n = 3 mice per genotype) and 6- to 8-mo-old (n = 3 mice per genotype) mice. Obviously, CSNK2B protein is absent in csnk2b ∆/∆ , HSA-Cre muscle lysates. ACTN2 served as loading control. (G and H) Graphs represent protein amounts of CSNK2 subunits which were analyzed before by western blot (F). Note, in response to the absence of Csnk2b [ 35 S]-radiolabeled yeast proteins Cox4, Mdh1 and Atp2 were individually imported into mitochondria (Δψ, membrane potential). Mitochondria were treated with proteinase K and analyzed by SDS-PAGE. p, precursor; m, mature. Import into mitochondria after the longest import time was set to 100% (control). Note, all mitochondrial precursor (p) proteins are imported into mitochondria and processed to shorter mature (m) size with similar time kinetic between control and the csnk2b ∆/∆ , HSA-Cre indicating mitochondrial protein import in vitro being unaffected. (J) Mitochondria were isolated from skeletal muscles of adult wild-type or csnk2b ∆/∆ , HSA-Cre mice and equal amounts of native TOMM complexes were resolved by blue-native gels (BN) which are used to separate native protein complexes. After western blot, different TOMM family members within native TOMM complexes were detected by specific antibodies as shown by representative images. The amount of these TOMM family members is similar between TOMM complexes from mitochondria of wild-type or csnk2b ∆/∆ , HSA-Cre muscles, indicating proper biogenesis of TOMM complexes.

    Article Snippet: Lysis of the tissue was performed by adding the same volume of a solution composed of 2 mg/ml proteinase K (Carl Roth, 7528.3), 0.5% sodium dodecyl sulfate (Carl Roth, CN30) and 10 mM EDTA, and incubating at 37°C for 2 h. The genomic DNA was precipitated with isopropanol, dissolved in H2 0, and subjected to quantitative RT-PCR with mouse-specific primers as listed below.

    Techniques: Activity Assay, Purification, In Vitro, Recombinant, Mutagenesis, Expressing, Transfection, Cell Culture, SDS Page, Western Blot, Incubation, Mouse Assay, Isolation

    Integrity of the mitochondrial inner membrane. a Mitochondrial protein steady state levels were analyzed by SDS-PAGE and western blotting with antibodies directed against the indicated proteins. b Isolated mitochondria suspended in potassium chloride based isolation buffer were treated with Proteinase K (Prot. K) and analyzed as described in panel ( a )

    Journal: Metabolomics

    Article Title: Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

    doi: 10.1007/s11306-018-1352-x

    Figure Lengend Snippet: Integrity of the mitochondrial inner membrane. a Mitochondrial protein steady state levels were analyzed by SDS-PAGE and western blotting with antibodies directed against the indicated proteins. b Isolated mitochondria suspended in potassium chloride based isolation buffer were treated with Proteinase K (Prot. K) and analyzed as described in panel ( a )

    Article Snippet: Proteinase K was inactivated by addition of 2.4 mM PMSF (Phenylmethylsulfonylfluorid, Roth) and further incubated on ice for 10 min. Mitochondria were pelleted for 10 min at 20,817× g (14,000 rpm, 5417R, Eppendorf) at 4 °C and washed with 200 µL isolation buffer containing 1 mM PMSF.

    Techniques: SDS Page, Western Blot, Isolation