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  • 99
    Thermo Fisher coomassie brilliant blue r 250
    CtBP2 forms a complex with p300 and Runx2 in vivo . (A) In vivo pull-down of the Flag-HA-CtBP2-associated complexes. The HOB-1 cells were transfected with pCDNA3-2xFlag-3xHA-CtBP2 or pCDNA3-2xFlag-3xHA . After 48 hours, the cells were lysed and subjected to IP analysis. The resulting protein complexes were loaded onto SDS-PAGE gel for separation and then stained with <t>Coomassie</t> Brilliant Blue R 250. The IgG and CtBP2 bands were indicated. (B) Verification of CtBP2-associated proteins identified in LC-MS/MS analysis. The protein samples used in (A) were used to verify the association of CtBP2, p300 and Runx2. (C and D) CtBP2 directly interacted with p300 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-p300 , pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc , or pCDNA3-2xFlag + pCDNA3-6xMyc-p300 . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (C) or anti-Myc-agarose (D) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures. (E and F) CtBP2 cannot directly interact with Runx2 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-Runx2 , pCDNA3-2xFlag + pCDNA3-6xMyc-Runx2 , or pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (E) or anti-Myc-agarose (F) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures.
    Coomassie Brilliant Blue R 250, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore coomassie brilliant blue r 250
    CtBP2 forms a complex with p300 and Runx2 in vivo . (A) In vivo pull-down of the Flag-HA-CtBP2-associated complexes. The HOB-1 cells were transfected with pCDNA3-2xFlag-3xHA-CtBP2 or pCDNA3-2xFlag-3xHA . After 48 hours, the cells were lysed and subjected to IP analysis. The resulting protein complexes were loaded onto SDS-PAGE gel for separation and then stained with <t>Coomassie</t> Brilliant Blue R 250. The IgG and CtBP2 bands were indicated. (B) Verification of CtBP2-associated proteins identified in LC-MS/MS analysis. The protein samples used in (A) were used to verify the association of CtBP2, p300 and Runx2. (C and D) CtBP2 directly interacted with p300 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-p300 , pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc , or pCDNA3-2xFlag + pCDNA3-6xMyc-p300 . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (C) or anti-Myc-agarose (D) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures. (E and F) CtBP2 cannot directly interact with Runx2 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-Runx2 , pCDNA3-2xFlag + pCDNA3-6xMyc-Runx2 , or pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (E) or anti-Myc-agarose (F) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures.
    Coomassie Brilliant Blue R 250, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad coomassie brilliant blue r 250
    Identification of XDH as a protein marker associated with the bulk of microRNAs in commercial milk. (a) Proteins extracted from the pellets obtained by ultracentrifugation of 100 mL of commercial milk were analysed by SDS-PAGE and <t>Coomassie</t> blue staining. The bands corresponding to those enriched in low-speed ultracentrifugation pellets were excised and submitted to LC-MS/MS analysis. The most enriched proteins in each pellet are displayed, based on the number of exclusive peptide count, percentage coverage of the protein and molecular weight match to the respective band. (b) The XDH protein content of the four ultracentrifugation pellets was assessed by Western blot (most representative of the three replicates, upper panel) and quantitative densitometry (lower panel) analyses (mean ± SD; n = 3). Western blot results are expressed as a percentage (%; mean ± SD) of the XDH proteins present in the four pellets. The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post-hoc comparison of the means with Tukey’s correction with p
    Coomassie Brilliant Blue R 250, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 4556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad coomassie brilliant blue r 250 staining solution
    Identification of XDH as a protein marker associated with the bulk of microRNAs in commercial milk. (a) Proteins extracted from the pellets obtained by ultracentrifugation of 100 mL of commercial milk were analysed by SDS-PAGE and <t>Coomassie</t> blue staining. The bands corresponding to those enriched in low-speed ultracentrifugation pellets were excised and submitted to LC-MS/MS analysis. The most enriched proteins in each pellet are displayed, based on the number of exclusive peptide count, percentage coverage of the protein and molecular weight match to the respective band. (b) The XDH protein content of the four ultracentrifugation pellets was assessed by Western blot (most representative of the three replicates, upper panel) and quantitative densitometry (lower panel) analyses (mean ± SD; n = 3). Western blot results are expressed as a percentage (%; mean ± SD) of the XDH proteins present in the four pellets. The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post-hoc comparison of the means with Tukey’s correction with p
    Coomassie Brilliant Blue R 250 Staining Solution, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 414 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad coomassie brilliant blue r250
    Identification of XDH as a protein marker associated with the bulk of microRNAs in commercial milk. (a) Proteins extracted from the pellets obtained by ultracentrifugation of 100 mL of commercial milk were analysed by SDS-PAGE and <t>Coomassie</t> blue staining. The bands corresponding to those enriched in low-speed ultracentrifugation pellets were excised and submitted to LC-MS/MS analysis. The most enriched proteins in each pellet are displayed, based on the number of exclusive peptide count, percentage coverage of the protein and molecular weight match to the respective band. (b) The XDH protein content of the four ultracentrifugation pellets was assessed by Western blot (most representative of the three replicates, upper panel) and quantitative densitometry (lower panel) analyses (mean ± SD; n = 3). Western blot results are expressed as a percentage (%; mean ± SD) of the XDH proteins present in the four pellets. The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post-hoc comparison of the means with Tukey’s correction with p
    Coomassie Brilliant Blue R250, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/coomassie brilliant blue r250/product/Bio-Rad
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    92
    Merck KGaA coomassie brilliant blue r 250
    Recombinant SARS-CoV-2 proteins. (A) Schematic drawing representing recombinantly expressed SARS-CoV-2 spike glycoprotein (S Strep , S His ), extended S1 subunit (S1e), receptor binding domain (RBD), and S2 subunit. (B) 4-12% Bis-Tris gel loaded with 5 μg of SARS-CoV-2 open reading frame (ORF) 8, S Strep , S His , S1e, RBD, S2, and human angiotensin converting enzyme 2 (hACE2) stained with <t>Coomassie</t> Brilliant Blue R 250. (C) Silver staining of 2 μg of SARS-CoV-2 S Strep . (D) Immunoblot of 2 μg of SARS-CoV-2 S Strep .
    Coomassie Brilliant Blue R 250, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco coomassie brilliant blue r 250
    Identification of S. pombe Atg38 and its AIM-mediated interaction with Atg8. (A) The domain organization of mouse NRBF2, budding yeast Atg38, and fission yeast SPBC660.08/Atg38. MIT, microtubule interacting and trafficking domain. CC, coiled-coil domain. AIM, Atg8-family-interacting motif. (B) Deletion of atg38 blocked starvation-induced processing of CFP-Atg8. Cells of wild type (WT) and mutants lacking PtdIns3K complexes subunits were collected before (+N) and after shifting to nitrogen-free medium for 10 h (−N), and the total lysates were analyzed by immunoblotting with antibody against CFP. (C) Atg38 colocalized with Atg8 at cytoplasmic puncta induced by starvation, and atg14Δ abolished the puncta of Atg38 and Atg8. Mid-log phase cells expressing YFP-tagged Atg38 and CFP-tagged Atg8 were incubated in nitrogen-free medium for 2 h, and then imaged by fluorescence microscopy. Arrowheads point to representative puncta where Atg38 and Atg8 colocalized. Scale bar: 3 μm. (D) The most conserved region of Atg38 among S. pombe, S. octosporus, S. cryophilus , and S. japonicus . (E-F) Coimmunoprecipitation between Atg38 and Atg8 was diminished by the AIM mutation in Atg38 (E) and the AIM-binding region mutation in Atg8 (F). Atg8 and Atg8 P52A,R67A were tagged with GFP. mCherry-tagged wild-type or AIM-mutated Atg38 was immunoprecipitated with mCherry-trap agarose beads. Total cell lysates and mCherry-trap precipitates were analyzed by immunoblotting with antibody against mCherry or GFP. (G) In yeast two-hybrid assay, Atg38 interacted with Atg8 in a manner dependent on the AIM. (H) An in vitro affinity-isolation assay between GST-tagged Atg38 fragments and HA-tagged Atg8 or Atg8 P52A,R67A . <t>Coomassie</t> Brilliant Blue R-250 (CBB) stained gel shows the GST construct inputs. Western blot was probed with antibody against HA
    Coomassie Brilliant Blue R 250, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific coomassie brilliant blue r 250
    Horizontal nondenaturing PAGE displaying migration of anionic (BSA) and cationic (lysozyme) proteins. PAGE was performed by loading 15 μL of 2 μg/μL protein solution mixed with 3μL 6X mixed dyes solution on the wells in the middle of a gel (5% Acrylamide-N,N-bisacrylamide, 38:2). After running with non-denaturing buffer (30 mM MOPS-25 mM histidine pH 6.5), at constant voltage (100V) for 1 hour, at 4 °C, the gel was stained with <t>Coomassie</t> Blue R-250 according to the protocol published by Dong et al. [ 5 ]. The figure displays migration of BSA (pI 4.8) toward the anode and migration of lysozyme (pI 11.3) toward the cathode.
    Coomassie Brilliant Blue R 250, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 91/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co coomassie brilliant blue r 250
    Horizontal nondenaturing PAGE displaying migration of anionic (BSA) and cationic (lysozyme) proteins. PAGE was performed by loading 15 μL of 2 μg/μL protein solution mixed with 3μL 6X mixed dyes solution on the wells in the middle of a gel (5% Acrylamide-N,N-bisacrylamide, 38:2). After running with non-denaturing buffer (30 mM MOPS-25 mM histidine pH 6.5), at constant voltage (100V) for 1 hour, at 4 °C, the gel was stained with <t>Coomassie</t> Blue R-250 according to the protocol published by Dong et al. [ 5 ]. The figure displays migration of BSA (pI 4.8) toward the anode and migration of lysozyme (pI 11.3) toward the cathode.
    Coomassie Brilliant Blue R 250, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM coomassie brilliant blue r 250
    HMGCS2 expression in a bacterial system. The purified GST protein was used as a negative control. (A) Western blotting of 600 ng purified wild-type and variant HMGCS2 expressed and purified from Escherichia coli and probed with anti-human HMGCS2 antibody. (B) Ponceau staining was performed on the same membrane used for the immunoblot and shows equal protein loading and transfer. (C) <t>Coomassie</t> brilliant blue R-250 staining of 450 ng of each sample shows equal distribution of proteins in the gel. (D) Specific enzymatic activity (dark gray bars) of wild-type and the G219E, M235T, V253A, S392L and R500C mutant variants of HMGCS2. Activity measurements were performed in three independent experiments. The error bars indicate standard deviations. Significant differences were observed compared to wild-type. * P
    Coomassie Brilliant Blue R 250, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nacalai coomassie brilliant blue r 250
    HMGCS2 expression in a bacterial system. The purified GST protein was used as a negative control. (A) Western blotting of 600 ng purified wild-type and variant HMGCS2 expressed and purified from Escherichia coli and probed with anti-human HMGCS2 antibody. (B) Ponceau staining was performed on the same membrane used for the immunoblot and shows equal protein loading and transfer. (C) <t>Coomassie</t> brilliant blue R-250 staining of 450 ng of each sample shows equal distribution of proteins in the gel. (D) Specific enzymatic activity (dark gray bars) of wild-type and the G219E, M235T, V253A, S392L and R500C mutant variants of HMGCS2. Activity measurements were performed in three independent experiments. The error bars indicate standard deviations. Significant differences were observed compared to wild-type. * P
    Coomassie Brilliant Blue R 250, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1 L Coomassie Brilliant Blue R 250 staining solution
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    Image Search Results


    CtBP2 forms a complex with p300 and Runx2 in vivo . (A) In vivo pull-down of the Flag-HA-CtBP2-associated complexes. The HOB-1 cells were transfected with pCDNA3-2xFlag-3xHA-CtBP2 or pCDNA3-2xFlag-3xHA . After 48 hours, the cells were lysed and subjected to IP analysis. The resulting protein complexes were loaded onto SDS-PAGE gel for separation and then stained with Coomassie Brilliant Blue R 250. The IgG and CtBP2 bands were indicated. (B) Verification of CtBP2-associated proteins identified in LC-MS/MS analysis. The protein samples used in (A) were used to verify the association of CtBP2, p300 and Runx2. (C and D) CtBP2 directly interacted with p300 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-p300 , pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc , or pCDNA3-2xFlag + pCDNA3-6xMyc-p300 . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (C) or anti-Myc-agarose (D) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures. (E and F) CtBP2 cannot directly interact with Runx2 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-Runx2 , pCDNA3-2xFlag + pCDNA3-6xMyc-Runx2 , or pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (E) or anti-Myc-agarose (F) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures.

    Journal: International Journal of Biological Sciences

    Article Title: The intracellular NADH level regulates atrophic nonunion pathogenesis through the CtBP2-p300-Runx2 transcriptional complex

    doi: 10.7150/ijbs.28302

    Figure Lengend Snippet: CtBP2 forms a complex with p300 and Runx2 in vivo . (A) In vivo pull-down of the Flag-HA-CtBP2-associated complexes. The HOB-1 cells were transfected with pCDNA3-2xFlag-3xHA-CtBP2 or pCDNA3-2xFlag-3xHA . After 48 hours, the cells were lysed and subjected to IP analysis. The resulting protein complexes were loaded onto SDS-PAGE gel for separation and then stained with Coomassie Brilliant Blue R 250. The IgG and CtBP2 bands were indicated. (B) Verification of CtBP2-associated proteins identified in LC-MS/MS analysis. The protein samples used in (A) were used to verify the association of CtBP2, p300 and Runx2. (C and D) CtBP2 directly interacted with p300 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-p300 , pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc , or pCDNA3-2xFlag + pCDNA3-6xMyc-p300 . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (C) or anti-Myc-agarose (D) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures. (E and F) CtBP2 cannot directly interact with Runx2 in HOB-1 cells. The HOB-1 cells were cotransfected with pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc-Runx2 , pCDNA3-2xFlag + pCDNA3-6xMyc-Runx2 , or pCDNA3-2xFlag-CtBP2 + pCDNA3-6xMyc . After 48 hours, the cells were lysed and subjected to IP analysis with either anti-Flag-agarose (E) or anti-Myc-agarose (F) . The pull-down products were then subjected to western blot with the antibodies indicated in the figures.

    Article Snippet: The resulting proteins were immunoprecipitated with anti-HA-agarose for at 4°C for 6 hours, and after washing five times with RIPA buffer, the HA-CtBP2 protein complex was loaded onto SDS-PAGE gels for electrophoresis, followed by staining with Coomassie Brilliant Blue R 250 (Thermo Fisher Scientific, #20278).

    Techniques: In Vivo, Transfection, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot

    Identification of XDH as a protein marker associated with the bulk of microRNAs in commercial milk. (a) Proteins extracted from the pellets obtained by ultracentrifugation of 100 mL of commercial milk were analysed by SDS-PAGE and Coomassie blue staining. The bands corresponding to those enriched in low-speed ultracentrifugation pellets were excised and submitted to LC-MS/MS analysis. The most enriched proteins in each pellet are displayed, based on the number of exclusive peptide count, percentage coverage of the protein and molecular weight match to the respective band. (b) The XDH protein content of the four ultracentrifugation pellets was assessed by Western blot (most representative of the three replicates, upper panel) and quantitative densitometry (lower panel) analyses (mean ± SD; n = 3). Western blot results are expressed as a percentage (%; mean ± SD) of the XDH proteins present in the four pellets. The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post-hoc comparison of the means with Tukey’s correction with p

    Journal: Journal of Extracellular Vesicles

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

    doi: 10.1080/20013078.2017.1401897

    Figure Lengend Snippet: Identification of XDH as a protein marker associated with the bulk of microRNAs in commercial milk. (a) Proteins extracted from the pellets obtained by ultracentrifugation of 100 mL of commercial milk were analysed by SDS-PAGE and Coomassie blue staining. The bands corresponding to those enriched in low-speed ultracentrifugation pellets were excised and submitted to LC-MS/MS analysis. The most enriched proteins in each pellet are displayed, based on the number of exclusive peptide count, percentage coverage of the protein and molecular weight match to the respective band. (b) The XDH protein content of the four ultracentrifugation pellets was assessed by Western blot (most representative of the three replicates, upper panel) and quantitative densitometry (lower panel) analyses (mean ± SD; n = 3). Western blot results are expressed as a percentage (%; mean ± SD) of the XDH proteins present in the four pellets. The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post-hoc comparison of the means with Tukey’s correction with p

    Article Snippet: After the run, the gels were stained with Coomassie blue (Coomassie Brilliant Blue R-250 Staining Solution; Bio-Rad Laboratories, Hercules, CA, USA; 0.2%) in 40% methanol and 10% acetic acid for 1 h and destained overnight with a destaining solution composed of 10% methanol and 10% glacial acetic acid.

    Techniques: Marker, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Molecular Weight, Western Blot

    Recombinant SARS-CoV-2 proteins. (A) Schematic drawing representing recombinantly expressed SARS-CoV-2 spike glycoprotein (S Strep , S His ), extended S1 subunit (S1e), receptor binding domain (RBD), and S2 subunit. (B) 4-12% Bis-Tris gel loaded with 5 μg of SARS-CoV-2 open reading frame (ORF) 8, S Strep , S His , S1e, RBD, S2, and human angiotensin converting enzyme 2 (hACE2) stained with Coomassie Brilliant Blue R 250. (C) Silver staining of 2 μg of SARS-CoV-2 S Strep . (D) Immunoblot of 2 μg of SARS-CoV-2 S Strep .

    Journal: bioRxiv

    Article Title: LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro

    doi: 10.1101/2020.12.02.408153

    Figure Lengend Snippet: Recombinant SARS-CoV-2 proteins. (A) Schematic drawing representing recombinantly expressed SARS-CoV-2 spike glycoprotein (S Strep , S His ), extended S1 subunit (S1e), receptor binding domain (RBD), and S2 subunit. (B) 4-12% Bis-Tris gel loaded with 5 μg of SARS-CoV-2 open reading frame (ORF) 8, S Strep , S His , S1e, RBD, S2, and human angiotensin converting enzyme 2 (hACE2) stained with Coomassie Brilliant Blue R 250. (C) Silver staining of 2 μg of SARS-CoV-2 S Strep . (D) Immunoblot of 2 μg of SARS-CoV-2 S Strep .

    Article Snippet: Proteins were visualized using Coomassie Brilliant Blue R 250 (Merck, Germany) or using the SilverQuest silver staining kit (Invitrogen Thermo Fisher Scientific, USA).

    Techniques: Recombinant, Binding Assay, Staining, Silver Staining

    Identification of S. pombe Atg38 and its AIM-mediated interaction with Atg8. (A) The domain organization of mouse NRBF2, budding yeast Atg38, and fission yeast SPBC660.08/Atg38. MIT, microtubule interacting and trafficking domain. CC, coiled-coil domain. AIM, Atg8-family-interacting motif. (B) Deletion of atg38 blocked starvation-induced processing of CFP-Atg8. Cells of wild type (WT) and mutants lacking PtdIns3K complexes subunits were collected before (+N) and after shifting to nitrogen-free medium for 10 h (−N), and the total lysates were analyzed by immunoblotting with antibody against CFP. (C) Atg38 colocalized with Atg8 at cytoplasmic puncta induced by starvation, and atg14Δ abolished the puncta of Atg38 and Atg8. Mid-log phase cells expressing YFP-tagged Atg38 and CFP-tagged Atg8 were incubated in nitrogen-free medium for 2 h, and then imaged by fluorescence microscopy. Arrowheads point to representative puncta where Atg38 and Atg8 colocalized. Scale bar: 3 μm. (D) The most conserved region of Atg38 among S. pombe, S. octosporus, S. cryophilus , and S. japonicus . (E-F) Coimmunoprecipitation between Atg38 and Atg8 was diminished by the AIM mutation in Atg38 (E) and the AIM-binding region mutation in Atg8 (F). Atg8 and Atg8 P52A,R67A were tagged with GFP. mCherry-tagged wild-type or AIM-mutated Atg38 was immunoprecipitated with mCherry-trap agarose beads. Total cell lysates and mCherry-trap precipitates were analyzed by immunoblotting with antibody against mCherry or GFP. (G) In yeast two-hybrid assay, Atg38 interacted with Atg8 in a manner dependent on the AIM. (H) An in vitro affinity-isolation assay between GST-tagged Atg38 fragments and HA-tagged Atg8 or Atg8 P52A,R67A . Coomassie Brilliant Blue R-250 (CBB) stained gel shows the GST construct inputs. Western blot was probed with antibody against HA

    Journal: Autophagy

    Article Title: Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop to promote autophagy

    doi: 10.1080/15548627.2020.1713644

    Figure Lengend Snippet: Identification of S. pombe Atg38 and its AIM-mediated interaction with Atg8. (A) The domain organization of mouse NRBF2, budding yeast Atg38, and fission yeast SPBC660.08/Atg38. MIT, microtubule interacting and trafficking domain. CC, coiled-coil domain. AIM, Atg8-family-interacting motif. (B) Deletion of atg38 blocked starvation-induced processing of CFP-Atg8. Cells of wild type (WT) and mutants lacking PtdIns3K complexes subunits were collected before (+N) and after shifting to nitrogen-free medium for 10 h (−N), and the total lysates were analyzed by immunoblotting with antibody against CFP. (C) Atg38 colocalized with Atg8 at cytoplasmic puncta induced by starvation, and atg14Δ abolished the puncta of Atg38 and Atg8. Mid-log phase cells expressing YFP-tagged Atg38 and CFP-tagged Atg8 were incubated in nitrogen-free medium for 2 h, and then imaged by fluorescence microscopy. Arrowheads point to representative puncta where Atg38 and Atg8 colocalized. Scale bar: 3 μm. (D) The most conserved region of Atg38 among S. pombe, S. octosporus, S. cryophilus , and S. japonicus . (E-F) Coimmunoprecipitation between Atg38 and Atg8 was diminished by the AIM mutation in Atg38 (E) and the AIM-binding region mutation in Atg8 (F). Atg8 and Atg8 P52A,R67A were tagged with GFP. mCherry-tagged wild-type or AIM-mutated Atg38 was immunoprecipitated with mCherry-trap agarose beads. Total cell lysates and mCherry-trap precipitates were analyzed by immunoblotting with antibody against mCherry or GFP. (G) In yeast two-hybrid assay, Atg38 interacted with Atg8 in a manner dependent on the AIM. (H) An in vitro affinity-isolation assay between GST-tagged Atg38 fragments and HA-tagged Atg8 or Atg8 P52A,R67A . Coomassie Brilliant Blue R-250 (CBB) stained gel shows the GST construct inputs. Western blot was probed with antibody against HA

    Article Snippet: The supernatants of cell lysates and incubated beads were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Amresco, 0472) or immunoblotted with an antibody against HA (MBL, M180-3).

    Techniques: Expressing, Incubation, Fluorescence, Microscopy, Mutagenesis, Binding Assay, Immunoprecipitation, Y2H Assay, In Vitro, Isolation, Staining, Construct, Western Blot

    Horizontal nondenaturing PAGE displaying migration of anionic (BSA) and cationic (lysozyme) proteins. PAGE was performed by loading 15 μL of 2 μg/μL protein solution mixed with 3μL 6X mixed dyes solution on the wells in the middle of a gel (5% Acrylamide-N,N-bisacrylamide, 38:2). After running with non-denaturing buffer (30 mM MOPS-25 mM histidine pH 6.5), at constant voltage (100V) for 1 hour, at 4 °C, the gel was stained with Coomassie Blue R-250 according to the protocol published by Dong et al. [ 5 ]. The figure displays migration of BSA (pI 4.8) toward the anode and migration of lysozyme (pI 11.3) toward the cathode.

    Journal: bioRxiv

    Article Title: Label-free horizontal EMSA for analysis of protein-RNA interactions

    doi: 10.1101/825679

    Figure Lengend Snippet: Horizontal nondenaturing PAGE displaying migration of anionic (BSA) and cationic (lysozyme) proteins. PAGE was performed by loading 15 μL of 2 μg/μL protein solution mixed with 3μL 6X mixed dyes solution on the wells in the middle of a gel (5% Acrylamide-N,N-bisacrylamide, 38:2). After running with non-denaturing buffer (30 mM MOPS-25 mM histidine pH 6.5), at constant voltage (100V) for 1 hour, at 4 °C, the gel was stained with Coomassie Blue R-250 according to the protocol published by Dong et al. [ 5 ]. The figure displays migration of BSA (pI 4.8) toward the anode and migration of lysozyme (pI 11.3) toward the cathode.

    Article Snippet: Coomassie Brilliant Blue R-250 was purchased from Fisher Scientific (Fair Lawn, NJ, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Migration, Staining

    HMGCS2 expression in a bacterial system. The purified GST protein was used as a negative control. (A) Western blotting of 600 ng purified wild-type and variant HMGCS2 expressed and purified from Escherichia coli and probed with anti-human HMGCS2 antibody. (B) Ponceau staining was performed on the same membrane used for the immunoblot and shows equal protein loading and transfer. (C) Coomassie brilliant blue R-250 staining of 450 ng of each sample shows equal distribution of proteins in the gel. (D) Specific enzymatic activity (dark gray bars) of wild-type and the G219E, M235T, V253A, S392L and R500C mutant variants of HMGCS2. Activity measurements were performed in three independent experiments. The error bars indicate standard deviations. Significant differences were observed compared to wild-type. * P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Japanese patients with mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: In vitro functional analysis of five novel HMGCS2 mutations

    doi: 10.3892/etm.2020.9166

    Figure Lengend Snippet: HMGCS2 expression in a bacterial system. The purified GST protein was used as a negative control. (A) Western blotting of 600 ng purified wild-type and variant HMGCS2 expressed and purified from Escherichia coli and probed with anti-human HMGCS2 antibody. (B) Ponceau staining was performed on the same membrane used for the immunoblot and shows equal protein loading and transfer. (C) Coomassie brilliant blue R-250 staining of 450 ng of each sample shows equal distribution of proteins in the gel. (D) Specific enzymatic activity (dark gray bars) of wild-type and the G219E, M235T, V253A, S392L and R500C mutant variants of HMGCS2. Activity measurements were performed in three independent experiments. The error bars indicate standard deviations. Significant differences were observed compared to wild-type. * P

    Article Snippet: In another experiment, 450 ng each sample was separated by 10% SDS-PAGE before the proteins in the gel were stained with Coomassie brilliant blue R-250 (cat. no. 031-17922; FUJIFILM Wako Pure Chemical Corporation) for 20 min at room temperature.

    Techniques: Expressing, Purification, Negative Control, Western Blot, Variant Assay, Staining, Activity Assay, Mutagenesis