Structured Review

Merck & Co proteinase k
Agarose gel electrophoresis on wild-type cells and hSMUG1 transfectants cultured with and without tetracycline. Whole cells were cast in agarose blocks, digested with <t>proteinase</t> K and subjected to electrophoresis. The gel was run at a low voltage, blotted and hybridised with a telomeric repeat probe. Subsequently, the membrane was stripped and hybridised with a probe for the α/β- tubulin gene array.
Proteinase K, supplied by Merck & Co, 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 "Expression of the human DNA glycosylase hSMUG1 in Trypanosoma brucei causes DNA damage and interferes with J biosynthesis"

Article Title: Expression of the human DNA glycosylase hSMUG1 in Trypanosoma brucei causes DNA damage and interferes with J biosynthesis

Journal: Nucleic Acids Research

doi:

Agarose gel electrophoresis on wild-type cells and hSMUG1 transfectants cultured with and without tetracycline. Whole cells were cast in agarose blocks, digested with proteinase K and subjected to electrophoresis. The gel was run at a low voltage, blotted and hybridised with a telomeric repeat probe. Subsequently, the membrane was stripped and hybridised with a probe for the α/β- tubulin gene array.
Figure Legend Snippet: Agarose gel electrophoresis on wild-type cells and hSMUG1 transfectants cultured with and without tetracycline. Whole cells were cast in agarose blocks, digested with proteinase K and subjected to electrophoresis. The gel was run at a low voltage, blotted and hybridised with a telomeric repeat probe. Subsequently, the membrane was stripped and hybridised with a probe for the α/β- tubulin gene array.

Techniques Used: Agarose Gel Electrophoresis, Cell Culture, Electrophoresis

2) Product Images from "Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis"

Article Title: Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0175810

Core protein envelopment analysis by membrane protection assay. Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/ΔE1E2 (ΔE1E2) or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, cells were lysed by freeze-thaw cycles and left untreated or treated with 5 μg/mL proteinase K (PK) in the presence or absence of 1% Triton X-100 (Triton). The samples were subjected to (A) western blot analysis with mAb against HCV core protein (a representative western blot is shown) or (B) dot blot for quantification of signal intensities with ImageJ (values were normalized to untreated samples). The mean values and standard errors of four independent experiments are shown. *, P comprised between 0.05 and 0.01; **, P comprised between 0.01 and 0.001; ***, P below 0.001.
Figure Legend Snippet: Core protein envelopment analysis by membrane protection assay. Huh-7.5.1 cells were transfected with the full-length HCV RNAs Con1/C3 (WT), Con1/C3/ΔE1E2 (ΔE1E2) or Con1/C3/Sp1mt (Sp1mt). Two days post-transfection, cells were lysed by freeze-thaw cycles and left untreated or treated with 5 μg/mL proteinase K (PK) in the presence or absence of 1% Triton X-100 (Triton). The samples were subjected to (A) western blot analysis with mAb against HCV core protein (a representative western blot is shown) or (B) dot blot for quantification of signal intensities with ImageJ (values were normalized to untreated samples). The mean values and standard errors of four independent experiments are shown. *, P comprised between 0.05 and 0.01; **, P comprised between 0.01 and 0.001; ***, P below 0.001.

Techniques Used: Transfection, Western Blot, Dot Blot

3) Product Images from "A novel and rapid method for obtaining high titre intact prion strains from mammalian brain"

Article Title: A novel and rapid method for obtaining high titre intact prion strains from mammalian brain

Journal: Scientific Reports

doi: 10.1038/srep10062

Overview of the purification method. ( a ) Western blot of 10% (w/v) terminal RML-infected CD1 mouse brain homogenate before (-) or after (+) digestion with proteinase K (PK) using anti-PrP monoclonal antibody ICSM35. ( b ) Purification scheme (SL, surface layer; P, pellet, SN, supernatant). ( c ) Silver-stained 16% SDS-PAGE gel showing RML-infected CD1 mouse brain homogenate (BH) and purified fractions. The equivalent of 2 μl or 200 μl (denoted by *) of 10% (w/v) brain homogenate was loaded per lane. +,– symbols indicate whether PK-digestion was applied during purification (to the P2 sample, see panel b).
Figure Legend Snippet: Overview of the purification method. ( a ) Western blot of 10% (w/v) terminal RML-infected CD1 mouse brain homogenate before (-) or after (+) digestion with proteinase K (PK) using anti-PrP monoclonal antibody ICSM35. ( b ) Purification scheme (SL, surface layer; P, pellet, SN, supernatant). ( c ) Silver-stained 16% SDS-PAGE gel showing RML-infected CD1 mouse brain homogenate (BH) and purified fractions. The equivalent of 2 μl or 200 μl (denoted by *) of 10% (w/v) brain homogenate was loaded per lane. +,– symbols indicate whether PK-digestion was applied during purification (to the P2 sample, see panel b).

Techniques Used: Purification, Western Blot, Infection, Staining, SDS Page

4) Product Images from "The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC"

Article Title: The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.9

Topology of the EAEC TssL protein. (A) Accessibility of cysteine residues. Whole EAEC tssL cells producing the Flag TssL or Flag TssL-Lt ( Flag TssL carrying a C-terminal CCPGCC motif) were treated (+) or not (−) with the 3-( N -)MPB probe, solubilized, and the TssL proteins were precipitated using agarose beads coupled to M2 anti-FLAG antibody. Precipitated material was subjected to SDS-PAGE and Western blot analysis using anti-FLAG antibody (to detect TssL, upper panel) and streptavidin coupled to alkaline phosphatase (to detect biotinylated TssL, lower panel). Molecular weight markers are indicated on the left. (B) Accessibility of TssL to proteinase K. Whole cells (WC), spheroplasts (Sph.), or purified membranes (Membr.) of tssL EAEC cells producing the Flag TssL protein were treated (+) or not (−) with proteinase K and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (an in-to-out topology inner membrane protein with the bulk of the protein in the periplasm, lower panel) and anti-FLAG (upper panel) antibodies. (C) Accessibility of TssL to CPY. Purified membranes of tssL EAEC cells producing the Flag TssL protein (TssL) or a Flag TssL protein fused to the 211-amino acid periplasmic domain of TagL (TssL-PG) were treated (+) or not (−) with CPY and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (lower panel) and anti-FLAG (upper panel) antibodies. (D) Topology model for the TssL protein at the inner membrane. The positions of the labeled (C-terminus) and unlabeled (C40, C68, C127, C129, and C200) cysteine residues are indicated by filled and open circles, respectively. The membrane boundaries of the transmembrane segment predicted by computer algorithms and identified by the accessibility studies are indicated.
Figure Legend Snippet: Topology of the EAEC TssL protein. (A) Accessibility of cysteine residues. Whole EAEC tssL cells producing the Flag TssL or Flag TssL-Lt ( Flag TssL carrying a C-terminal CCPGCC motif) were treated (+) or not (−) with the 3-( N -)MPB probe, solubilized, and the TssL proteins were precipitated using agarose beads coupled to M2 anti-FLAG antibody. Precipitated material was subjected to SDS-PAGE and Western blot analysis using anti-FLAG antibody (to detect TssL, upper panel) and streptavidin coupled to alkaline phosphatase (to detect biotinylated TssL, lower panel). Molecular weight markers are indicated on the left. (B) Accessibility of TssL to proteinase K. Whole cells (WC), spheroplasts (Sph.), or purified membranes (Membr.) of tssL EAEC cells producing the Flag TssL protein were treated (+) or not (−) with proteinase K and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (an in-to-out topology inner membrane protein with the bulk of the protein in the periplasm, lower panel) and anti-FLAG (upper panel) antibodies. (C) Accessibility of TssL to CPY. Purified membranes of tssL EAEC cells producing the Flag TssL protein (TssL) or a Flag TssL protein fused to the 211-amino acid periplasmic domain of TagL (TssL-PG) were treated (+) or not (−) with CPY and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (lower panel) and anti-FLAG (upper panel) antibodies. (D) Topology model for the TssL protein at the inner membrane. The positions of the labeled (C-terminus) and unlabeled (C40, C68, C127, C129, and C200) cysteine residues are indicated by filled and open circles, respectively. The membrane boundaries of the transmembrane segment predicted by computer algorithms and identified by the accessibility studies are indicated.

Techniques Used: SDS Page, Western Blot, Molecular Weight, Purification, Labeling

5) Product Images from "Multiple SecA Molecules Drive Protein Translocation across a Single Translocon with SecG Inversion *"

Article Title: Multiple SecA Molecules Drive Protein Translocation across a Single Translocon with SecG Inversion *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.301754

Translocation intermediates of proOmpA translocation. A , [ 35 S]proOmpA translocation into IMV prepared from K003 was carried out at 37 °C in the presence of 60 μg/ml WT SecA ( upper left ), D209N SecA ( lower left ), or both (60 μg/ml each, upper right ). 15 min after the D209N SecA reaction had been initiated, WT SecA (60 μg/ml) was added and the reaction was continued at 37 °C for the periods indicated in parentheses ( lower right ). At each indicated time, an aliquot (25 μl) was withdrawn and chilled on ice to stop the reaction, followed by proteinase K treatment (1 mg/ml). The samples were analyzed by SDS-PAGE and autoradiography. The indicated amounts of proOmpA that had not been digested were also determined. The positions of proOmpA and mature OmpA are indicated by arrows denoted by p and m , respectively. The positions of the translocation intermediates are also indicated by arrows denoted by I 29 , I 28 , I 26 , and I 18 , respectively. B , the translocation activity of each sample in A at 10 min was quantitated. Black , gray , and white bars correspond to m, I 28 , and I 18 , respectively. The numbers of Met and Cys in OmpA, I 28 , and I 18 were regarded as 7, 4, and 4, respectively, on the basis of their molecular weights and amino acid sequences. C , 125 I WT SecA can insert into D209N SecA-saturated IMV. The translocation reaction for IMV prepared from K003 was carried out in the presence of 6 μg/ml D209N SecA and 25 μg/ml proOmpA at 37 °C for 10 min, and then either 125 I WT SecA ( left ) or 125 I D209N SecA ( right ) was added at 0.4 μg/ml, followed by further incubation at 37 °C. At each indicated time after the labeled SecA was added, an aliquot (100 μl) was withdrawn and chilled on ice, followed by proteinase K digestion at 1 mg/ml. Samples were then analyzed by SDS-PAGE and autoradiography. The sample from each reaction that had not been digested was also analyzed (- PK ). The positions of SecA and the membrane-protected fragment of 30 kDa are indicated by arrows .
Figure Legend Snippet: Translocation intermediates of proOmpA translocation. A , [ 35 S]proOmpA translocation into IMV prepared from K003 was carried out at 37 °C in the presence of 60 μg/ml WT SecA ( upper left ), D209N SecA ( lower left ), or both (60 μg/ml each, upper right ). 15 min after the D209N SecA reaction had been initiated, WT SecA (60 μg/ml) was added and the reaction was continued at 37 °C for the periods indicated in parentheses ( lower right ). At each indicated time, an aliquot (25 μl) was withdrawn and chilled on ice to stop the reaction, followed by proteinase K treatment (1 mg/ml). The samples were analyzed by SDS-PAGE and autoradiography. The indicated amounts of proOmpA that had not been digested were also determined. The positions of proOmpA and mature OmpA are indicated by arrows denoted by p and m , respectively. The positions of the translocation intermediates are also indicated by arrows denoted by I 29 , I 28 , I 26 , and I 18 , respectively. B , the translocation activity of each sample in A at 10 min was quantitated. Black , gray , and white bars correspond to m, I 28 , and I 18 , respectively. The numbers of Met and Cys in OmpA, I 28 , and I 18 were regarded as 7, 4, and 4, respectively, on the basis of their molecular weights and amino acid sequences. C , 125 I WT SecA can insert into D209N SecA-saturated IMV. The translocation reaction for IMV prepared from K003 was carried out in the presence of 6 μg/ml D209N SecA and 25 μg/ml proOmpA at 37 °C for 10 min, and then either 125 I WT SecA ( left ) or 125 I D209N SecA ( right ) was added at 0.4 μg/ml, followed by further incubation at 37 °C. At each indicated time after the labeled SecA was added, an aliquot (100 μl) was withdrawn and chilled on ice, followed by proteinase K digestion at 1 mg/ml. Samples were then analyzed by SDS-PAGE and autoradiography. The sample from each reaction that had not been digested was also analyzed (- PK ). The positions of SecA and the membrane-protected fragment of 30 kDa are indicated by arrows .

Techniques Used: Translocation Assay, SDS Page, Autoradiography, Activity Assay, Incubation, Labeling

6) Product Images from "Folding of Active ?-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum"

Article Title: Folding of Active ?-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

Journal: Molecular Biology of the Cell

doi:

Trypsin sensitivity of  35 S-Hsp150Δ-β-lactamase. (A)  Sec18–1  cells (5 × 10 8 /ml) were preincubated for 15 min at 37°C and  35 S labeled for 30 min. (B)  Sec62–101  cells (5 × 10 8 /ml) were preincubated for 15 min at 30°C and labeled for 5 min. The cells were lysed under nondenaturing conditions and immunoprecipitated with anti-β-lactamase antiserum. The precipitates were divided in equal portions and digested with trypsin or proteinase K, as indicated, and analyzed by SDS-PAGE. The β-lactamase-related proteins (kDa) are indicated on the right and the molecular weight markers (kDa) on the left.
Figure Legend Snippet: Trypsin sensitivity of 35 S-Hsp150Δ-β-lactamase. (A) Sec18–1 cells (5 × 10 8 /ml) were preincubated for 15 min at 37°C and 35 S labeled for 30 min. (B) Sec62–101 cells (5 × 10 8 /ml) were preincubated for 15 min at 30°C and labeled for 5 min. The cells were lysed under nondenaturing conditions and immunoprecipitated with anti-β-lactamase antiserum. The precipitates were divided in equal portions and digested with trypsin or proteinase K, as indicated, and analyzed by SDS-PAGE. The β-lactamase-related proteins (kDa) are indicated on the right and the molecular weight markers (kDa) on the left.

Techniques Used: Labeling, Immunoprecipitation, SDS Page, Molecular Weight

Trypsin sensitivity of microsome-associated Hsp150Δ-β-lactamase. Sec18–1 cells were incubated for 2 h at 37°C under high cell density conditions, followed by isolation of microsomes. The microsomal preparation was divided in equal portions, which received trypsin (TRY), proteinase K (pK), and Triton X-100 (TX-100), as indicated. The digests were subjected to SDS-PAGE in 7.5–15% gels and Western blotting using anti-β-lactamase antiserum. The β-lactamase-related proteins (kDa) are indicated.
Figure Legend Snippet: Trypsin sensitivity of microsome-associated Hsp150Δ-β-lactamase. Sec18–1 cells were incubated for 2 h at 37°C under high cell density conditions, followed by isolation of microsomes. The microsomal preparation was divided in equal portions, which received trypsin (TRY), proteinase K (pK), and Triton X-100 (TX-100), as indicated. The digests were subjected to SDS-PAGE in 7.5–15% gels and Western blotting using anti-β-lactamase antiserum. The β-lactamase-related proteins (kDa) are indicated.

Techniques Used: Incubation, Isolation, SDS Page, Western Blot

7) Product Images from "Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1"

Article Title: Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

Journal: Experimental Cell Research

doi: 10.1016/j.yexcr.2010.01.005

Differences in OPA1 levels are due to protein accessibility. (A) Semiquantitative RT-PCR was performed on mRNA isolated from Omi/HtrA2 WT or KO and re-transfected MEF cells. There are no differences in the OPA1 mRNA levels (upper panel). Amplification of β-actin served as loading control (lower panel). (B) For analysis of the full cell content, 150,000 control and Omi/HtrA2 KO MEF cells were harvested, lysed directly in Laemmli SDS-PAGE sample buffer and analyzed by Western blot. Immunoblots were probed with anti-OPA1 (short and long exposure) and anti-Omi/HtrA2 and β-actin was used as a loading control. (C) Mitochondrial and cytosolic fractions prepared from Omi/HtrA2 WT and KO cells were subjected to Western blotting and analyzed for localization of both OPA1 and Omi/HtrA2. The purity of the cytosolic (Cyto) and mitochondrial (Mito) fractions was assessed by probing for Hsp90 and VDAC1, respectively. (D) Mitochondria isolated from control (left lanes) or Omi/HtrA2 KO (right lanes) MEF cells were subjected to proteinase K digestion for indicated time points. Western blots were prepared and probed with anti-OPA1 and anti-Omi/HtrA2 as indicated. The outer membrane associated protein Fis1 was rapidly degraded whereas the inner membrane protein prohibitin was shielded. The higher molecular weight OPA1 band densitometrically analyzed (arrow head) is indicated and the fold change compared to untreated (time point 0) is shown below the Western blot (D).
Figure Legend Snippet: Differences in OPA1 levels are due to protein accessibility. (A) Semiquantitative RT-PCR was performed on mRNA isolated from Omi/HtrA2 WT or KO and re-transfected MEF cells. There are no differences in the OPA1 mRNA levels (upper panel). Amplification of β-actin served as loading control (lower panel). (B) For analysis of the full cell content, 150,000 control and Omi/HtrA2 KO MEF cells were harvested, lysed directly in Laemmli SDS-PAGE sample buffer and analyzed by Western blot. Immunoblots were probed with anti-OPA1 (short and long exposure) and anti-Omi/HtrA2 and β-actin was used as a loading control. (C) Mitochondrial and cytosolic fractions prepared from Omi/HtrA2 WT and KO cells were subjected to Western blotting and analyzed for localization of both OPA1 and Omi/HtrA2. The purity of the cytosolic (Cyto) and mitochondrial (Mito) fractions was assessed by probing for Hsp90 and VDAC1, respectively. (D) Mitochondria isolated from control (left lanes) or Omi/HtrA2 KO (right lanes) MEF cells were subjected to proteinase K digestion for indicated time points. Western blots were prepared and probed with anti-OPA1 and anti-Omi/HtrA2 as indicated. The outer membrane associated protein Fis1 was rapidly degraded whereas the inner membrane protein prohibitin was shielded. The higher molecular weight OPA1 band densitometrically analyzed (arrow head) is indicated and the fold change compared to untreated (time point 0) is shown below the Western blot (D).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Amplification, SDS Page, Western Blot, Molecular Weight

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Article Title: COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids
Article Snippet: DNA extraction was performed using Mole tissue kit on the Mole instrument (Mole Genetics, Lysaker, Norway; discontinued). .. Briefly, fin material was rinsed with distilled water and approximately 20 mg for each individual was transferred to a tube containing 100 μl Mole lysis buffer and 2 μl proteinase K from a 20 mg ml-1 stock solution (Merck, EC 3.4.21.14; www.merck.com ). .. The samples were incubated at 65°C for 1 h and further processed using the Mole instrument according to the manufacturer’s instructions.

Incubation:

Article Title: Visualization of individual DNA loops and a map of loop domains in the human dystrophin gene
Article Snippet: To cleave DNA by topoisomerase II in vivo , agarose blocks with embedded living cells were incubated for 30 min at 37°C in RPMI 1640 medium supplemented with VM-26 (10–50 µg/ml) as described ( ). .. In both cases, after incubation with VM-26 the agarose blocks were placed into stop buffer [1% SDS, 0.4 M EDTA (pH 8.0), 0.5 mg/ml proteinase K (Merck)] and digestion was carried out for 36 h at 55°C with constant gentle rotation. ..

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    Merck & Co proteinase k
    Differences in OPA1 levels are due to protein accessibility. (A) Semiquantitative RT-PCR was performed on mRNA isolated from Omi/HtrA2 WT or KO and re-transfected MEF cells. There are no differences in the OPA1 mRNA levels (upper panel). Amplification of β-actin served as loading control (lower panel). (B) For analysis of the full cell content, 150,000 control and Omi/HtrA2 KO MEF cells were harvested, lysed directly in Laemmli SDS-PAGE sample buffer and analyzed by Western blot. Immunoblots were probed with anti-OPA1 (short and long exposure) and anti-Omi/HtrA2 and β-actin was used as a loading control. (C) Mitochondrial and cytosolic fractions prepared from Omi/HtrA2 WT and KO cells were subjected to Western blotting and analyzed for localization of both OPA1 and Omi/HtrA2. The purity of the cytosolic (Cyto) and mitochondrial (Mito) fractions was assessed by probing for Hsp90 and VDAC1, respectively. (D) Mitochondria isolated from control (left lanes) or Omi/HtrA2 KO (right lanes) MEF cells were subjected to <t>proteinase</t> K digestion for indicated time points. Western blots were prepared and probed with anti-OPA1 and anti-Omi/HtrA2 as indicated. The outer membrane associated protein Fis1 was rapidly degraded whereas the inner membrane protein prohibitin was shielded. The higher molecular weight OPA1 band densitometrically analyzed (arrow head) is indicated and the fold change compared to untreated (time point 0) is shown below the Western blot (D).
    Proteinase K, supplied by Merck & Co, 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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    Differences in OPA1 levels are due to protein accessibility. (A) Semiquantitative RT-PCR was performed on mRNA isolated from Omi/HtrA2 WT or KO and re-transfected MEF cells. There are no differences in the OPA1 mRNA levels (upper panel). Amplification of β-actin served as loading control (lower panel). (B) For analysis of the full cell content, 150,000 control and Omi/HtrA2 KO MEF cells were harvested, lysed directly in Laemmli SDS-PAGE sample buffer and analyzed by Western blot. Immunoblots were probed with anti-OPA1 (short and long exposure) and anti-Omi/HtrA2 and β-actin was used as a loading control. (C) Mitochondrial and cytosolic fractions prepared from Omi/HtrA2 WT and KO cells were subjected to Western blotting and analyzed for localization of both OPA1 and Omi/HtrA2. The purity of the cytosolic (Cyto) and mitochondrial (Mito) fractions was assessed by probing for Hsp90 and VDAC1, respectively. (D) Mitochondria isolated from control (left lanes) or Omi/HtrA2 KO (right lanes) MEF cells were subjected to proteinase K digestion for indicated time points. Western blots were prepared and probed with anti-OPA1 and anti-Omi/HtrA2 as indicated. The outer membrane associated protein Fis1 was rapidly degraded whereas the inner membrane protein prohibitin was shielded. The higher molecular weight OPA1 band densitometrically analyzed (arrow head) is indicated and the fold change compared to untreated (time point 0) is shown below the Western blot (D).

    Journal: Experimental Cell Research

    Article Title: Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

    doi: 10.1016/j.yexcr.2010.01.005

    Figure Lengend Snippet: Differences in OPA1 levels are due to protein accessibility. (A) Semiquantitative RT-PCR was performed on mRNA isolated from Omi/HtrA2 WT or KO and re-transfected MEF cells. There are no differences in the OPA1 mRNA levels (upper panel). Amplification of β-actin served as loading control (lower panel). (B) For analysis of the full cell content, 150,000 control and Omi/HtrA2 KO MEF cells were harvested, lysed directly in Laemmli SDS-PAGE sample buffer and analyzed by Western blot. Immunoblots were probed with anti-OPA1 (short and long exposure) and anti-Omi/HtrA2 and β-actin was used as a loading control. (C) Mitochondrial and cytosolic fractions prepared from Omi/HtrA2 WT and KO cells were subjected to Western blotting and analyzed for localization of both OPA1 and Omi/HtrA2. The purity of the cytosolic (Cyto) and mitochondrial (Mito) fractions was assessed by probing for Hsp90 and VDAC1, respectively. (D) Mitochondria isolated from control (left lanes) or Omi/HtrA2 KO (right lanes) MEF cells were subjected to proteinase K digestion for indicated time points. Western blots were prepared and probed with anti-OPA1 and anti-Omi/HtrA2 as indicated. The outer membrane associated protein Fis1 was rapidly degraded whereas the inner membrane protein prohibitin was shielded. The higher molecular weight OPA1 band densitometrically analyzed (arrow head) is indicated and the fold change compared to untreated (time point 0) is shown below the Western blot (D).

    Article Snippet: For proteinase K digest, 20 μg mitochondria were subjected to 20 ng proteinase K (Merck, EC 3.4.21.64) digestion at 37 °C for 0, 5, 10, 20, and 40 min.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Amplification, SDS Page, Western Blot, Molecular Weight

    Topology of the EAEC TssL protein. (A) Accessibility of cysteine residues. Whole EAEC tssL cells producing the Flag TssL or Flag TssL-Lt ( Flag TssL carrying a C-terminal CCPGCC motif) were treated (+) or not (−) with the 3-( N -)MPB probe, solubilized, and the TssL proteins were precipitated using agarose beads coupled to M2 anti-FLAG antibody. Precipitated material was subjected to SDS-PAGE and Western blot analysis using anti-FLAG antibody (to detect TssL, upper panel) and streptavidin coupled to alkaline phosphatase (to detect biotinylated TssL, lower panel). Molecular weight markers are indicated on the left. (B) Accessibility of TssL to proteinase K. Whole cells (WC), spheroplasts (Sph.), or purified membranes (Membr.) of tssL EAEC cells producing the Flag TssL protein were treated (+) or not (−) with proteinase K and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (an in-to-out topology inner membrane protein with the bulk of the protein in the periplasm, lower panel) and anti-FLAG (upper panel) antibodies. (C) Accessibility of TssL to CPY. Purified membranes of tssL EAEC cells producing the Flag TssL protein (TssL) or a Flag TssL protein fused to the 211-amino acid periplasmic domain of TagL (TssL-PG) were treated (+) or not (−) with CPY and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (lower panel) and anti-FLAG (upper panel) antibodies. (D) Topology model for the TssL protein at the inner membrane. The positions of the labeled (C-terminus) and unlabeled (C40, C68, C127, C129, and C200) cysteine residues are indicated by filled and open circles, respectively. The membrane boundaries of the transmembrane segment predicted by computer algorithms and identified by the accessibility studies are indicated.

    Journal: MicrobiologyOpen

    Article Title: The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC

    doi: 10.1002/mbo3.9

    Figure Lengend Snippet: Topology of the EAEC TssL protein. (A) Accessibility of cysteine residues. Whole EAEC tssL cells producing the Flag TssL or Flag TssL-Lt ( Flag TssL carrying a C-terminal CCPGCC motif) were treated (+) or not (−) with the 3-( N -)MPB probe, solubilized, and the TssL proteins were precipitated using agarose beads coupled to M2 anti-FLAG antibody. Precipitated material was subjected to SDS-PAGE and Western blot analysis using anti-FLAG antibody (to detect TssL, upper panel) and streptavidin coupled to alkaline phosphatase (to detect biotinylated TssL, lower panel). Molecular weight markers are indicated on the left. (B) Accessibility of TssL to proteinase K. Whole cells (WC), spheroplasts (Sph.), or purified membranes (Membr.) of tssL EAEC cells producing the Flag TssL protein were treated (+) or not (−) with proteinase K and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (an in-to-out topology inner membrane protein with the bulk of the protein in the periplasm, lower panel) and anti-FLAG (upper panel) antibodies. (C) Accessibility of TssL to CPY. Purified membranes of tssL EAEC cells producing the Flag TssL protein (TssL) or a Flag TssL protein fused to the 211-amino acid periplasmic domain of TagL (TssL-PG) were treated (+) or not (−) with CPY and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (lower panel) and anti-FLAG (upper panel) antibodies. (D) Topology model for the TssL protein at the inner membrane. The positions of the labeled (C-terminus) and unlabeled (C40, C68, C127, C129, and C200) cysteine residues are indicated by filled and open circles, respectively. The membrane boundaries of the transmembrane segment predicted by computer algorithms and identified by the accessibility studies are indicated.

    Article Snippet: Sodium lauroyl sarcosinate (SLS), L-arabinose, N -ethyl-maleimide (NEM), and yeast carboxypeptidase Y (CPY) were purchased from Sigma-Aldrich (Saint-Quentin Falavier, France), AHT (used at 0.2 μg mL−1 throughout the study) from IBA, proteinase K from Merck, and 3-(N -maleimidyl-propionyl) biocytin (MPB) from Molecular probes.

    Techniques: SDS Page, Western Blot, Molecular Weight, Purification, Labeling

    Trypsin sensitivity of  35 S-Hsp150Δ-β-lactamase. (A)  Sec18–1  cells (5 × 10 8 /ml) were preincubated for 15 min at 37°C and  35 S labeled for 30 min. (B)  Sec62–101  cells (5 × 10 8 /ml) were preincubated for 15 min at 30°C and labeled for 5 min. The cells were lysed under nondenaturing conditions and immunoprecipitated with anti-β-lactamase antiserum. The precipitates were divided in equal portions and digested with trypsin or proteinase K, as indicated, and analyzed by SDS-PAGE. The β-lactamase-related proteins (kDa) are indicated on the right and the molecular weight markers (kDa) on the left.

    Journal: Molecular Biology of the Cell

    Article Title: Folding of Active ?-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: Trypsin sensitivity of 35 S-Hsp150Δ-β-lactamase. (A) Sec18–1 cells (5 × 10 8 /ml) were preincubated for 15 min at 37°C and 35 S labeled for 30 min. (B) Sec62–101 cells (5 × 10 8 /ml) were preincubated for 15 min at 30°C and labeled for 5 min. The cells were lysed under nondenaturing conditions and immunoprecipitated with anti-β-lactamase antiserum. The precipitates were divided in equal portions and digested with trypsin or proteinase K, as indicated, and analyzed by SDS-PAGE. The β-lactamase-related proteins (kDa) are indicated on the right and the molecular weight markers (kDa) on the left.

    Article Snippet: Isolated unlabeled microsomes were digested with trypsin (Sigma; 25 μg/ml) for 30 min at 10°C, followed by addition of phenylmethylsulfonyl fluoride to the final concentration of 1 mM and heating in a boiling water bath for 5 min. Proteinase K (Merck, St. Louis, MO; 16 μg/ml) digestion occurred for 90 min at 0°C.

    Techniques: Labeling, Immunoprecipitation, SDS Page, Molecular Weight

    Trypsin sensitivity of microsome-associated Hsp150Δ-β-lactamase. Sec18–1 cells were incubated for 2 h at 37°C under high cell density conditions, followed by isolation of microsomes. The microsomal preparation was divided in equal portions, which received trypsin (TRY), proteinase K (pK), and Triton X-100 (TX-100), as indicated. The digests were subjected to SDS-PAGE in 7.5–15% gels and Western blotting using anti-β-lactamase antiserum. The β-lactamase-related proteins (kDa) are indicated.

    Journal: Molecular Biology of the Cell

    Article Title: Folding of Active ?-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: Trypsin sensitivity of microsome-associated Hsp150Δ-β-lactamase. Sec18–1 cells were incubated for 2 h at 37°C under high cell density conditions, followed by isolation of microsomes. The microsomal preparation was divided in equal portions, which received trypsin (TRY), proteinase K (pK), and Triton X-100 (TX-100), as indicated. The digests were subjected to SDS-PAGE in 7.5–15% gels and Western blotting using anti-β-lactamase antiserum. The β-lactamase-related proteins (kDa) are indicated.

    Article Snippet: Isolated unlabeled microsomes were digested with trypsin (Sigma; 25 μg/ml) for 30 min at 10°C, followed by addition of phenylmethylsulfonyl fluoride to the final concentration of 1 mM and heating in a boiling water bath for 5 min. Proteinase K (Merck, St. Louis, MO; 16 μg/ml) digestion occurred for 90 min at 0°C.

    Techniques: Incubation, Isolation, SDS Page, Western Blot