mops sds running buffer  (Thermo Fisher)


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    Name:
    NuPAGE MOPS SDS Running Buffer 20X
    Description:
    NuPAGE MOPS SDS Running Buffer 20X is formulated for running proteins on NuPAGE Novex Bis Tris gels only NuPAGE MOPS SDS Running Buffer is recommended for separating medium to large sized proteins Use the right buffer to optimize protein separationsNuPAGE MES SDS Running Buffer and NuPAGE MOPS SDS Running Buffer can both be used with NuPAGE Novex Bis Tris gels Use of MOPS buffer allows proteins to run slower than when using MES buffer Get consistent convenient high quality resultsPre mixed NuPAGE buffers are a convenient way to ensure high quality consistent electrophoresis results All buffers are made with high purity reagents and are strictly quality controlled The buffers are provided as a concentrated solution and require simple dilution with deionized water before use Related links• Compare protein migration patterns with different combinations of gels and running buffers • Find NuPAGE Novex Bis Tris gels
    Catalog Number:
    np0001
    Price:
    None
    Applications:
    1D Gel Electrophoresis|NuPAGE® Novex® Bis-Tris Gel Electrophoresis|Protein Biology|Protein Gel Electrophoresis
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher mops sds running buffer
    CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% <t>SDS-PAGE),</t> using NuPAGE Bis-Tris gels with <t>MOPS-SDS</t> Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p
    NuPAGE MOPS SDS Running Buffer 20X is formulated for running proteins on NuPAGE Novex Bis Tris gels only NuPAGE MOPS SDS Running Buffer is recommended for separating medium to large sized proteins Use the right buffer to optimize protein separationsNuPAGE MES SDS Running Buffer and NuPAGE MOPS SDS Running Buffer can both be used with NuPAGE Novex Bis Tris gels Use of MOPS buffer allows proteins to run slower than when using MES buffer Get consistent convenient high quality resultsPre mixed NuPAGE buffers are a convenient way to ensure high quality consistent electrophoresis results All buffers are made with high purity reagents and are strictly quality controlled The buffers are provided as a concentrated solution and require simple dilution with deionized water before use Related links• Compare protein migration patterns with different combinations of gels and running buffers • Find NuPAGE Novex Bis Tris gels
    https://www.bioz.com/result/mops sds running buffer/product/Thermo Fisher
    Average 99 stars, based on 60 article reviews
    Price from $9.99 to $1999.99
    mops sds running buffer - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Role of microRNAs in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas"

    Article Title: Role of microRNAs in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7434

    CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p
    Figure Legend Snippet: CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p

    Techniques Used: Expressing, Transfection, Western Blot, Isolation, Polyacrylamide Gel Electrophoresis, SDS Page

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells . Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells
    Article Snippet: .. Protein samples were mixed with NuPAGE sample buffer containing dithiothreitol (DTT) and proceeded for SDS‐PAGE gel electrophoresis (NuPAGE precast 10% or 4–12% Bis‐tris gels in NuPAGE MOPS SDS running buffer, Invitrogen, Waltham, MA) in NOVEX chambers under reducing/denaturing conditions. .. To indicate the molecular weight of proteins we used a benchmark protein ladder (Invitrogen).

    Chromatography:

    Article Title: Biochemical Analysis of Protein SUMOylation
    Article Snippet: .. Luria-Bertani (LB) broth (Sigma), (20g LB powder/1 L H2 O), autoclaved 500 mg/ml Kanamycin stock solution (Fisher Scientific), 0.22 um filter-sterilized 100 mg/ml Ampicillin stock solution (Fisher Scientific), 0.22 um filter-sterilized 50 mg/ml Chloroamphenicol stock solution (Fisher Scientific) in ethanol, 0.22 um filter-sterilized 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientifics) Milli-Q-water Shaker Incubator (Thermo-Electron Corp.) Ultracentrifuge (Beckman Avanti J-25I) Spectramax M5 (Molecular Devices) BugBuster 10X protein extraction reagent (Novagen) Protease inhibitor cocktail (Roche) 2X Binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM Imidazole, and 0.04% NaN3 ) 2X GST binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl and 0.04% NaN3 ) Benzonase (Novagen) 50 mM Phenylmethylsulfonyl fluoride (PMSF) Dithiothreitol (DTT) (Sigma) Chromatography column (10x100 mm) (Sigma) Column stand with clamp Ni-NTA resin (Qiagen) Glutathione agarose resin (Amersham Biosciences) Reduced glutathione (Sigma) Wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM Imidazole) Elution buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM Imidazole) 1X GST wash buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) GST elution buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM Glutathione reduced) Dialysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT) Phosphate buffered saline (PBS) 15 ml 10kD and 30kD Centricon (Millipore) Cold room 2X SDS sample buffer with DTT 4–12% Bis-Tris gradient gel (Invitrogen) NuPAGE MOPS SDS running buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) SimplyBlue Stain (Invitrogen) 1.5 ml Sterile microcentrifuge tubes Bradford reagent (Bio-rad) Bradford reagent (Bio-rad) .. 1 Culture a single colony of freshly transformed cells in 25 ml of LB broth containing 0.5 mg/ml kanamycin and 0.05 mg/ml chloroamphenicol at 225 rpm for 12 hrs at 37 ºC. (For E1: LB broth contains 0.5 mg/ml kanamycin, 0.05 mg/ml chloroamphenicol and 0.1 mg/ml ampicillin).

    Protease Inhibitor:

    Article Title: Biochemical Analysis of Protein SUMOylation
    Article Snippet: .. Luria-Bertani (LB) broth (Sigma), (20g LB powder/1 L H2 O), autoclaved 500 mg/ml Kanamycin stock solution (Fisher Scientific), 0.22 um filter-sterilized 100 mg/ml Ampicillin stock solution (Fisher Scientific), 0.22 um filter-sterilized 50 mg/ml Chloroamphenicol stock solution (Fisher Scientific) in ethanol, 0.22 um filter-sterilized 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientifics) Milli-Q-water Shaker Incubator (Thermo-Electron Corp.) Ultracentrifuge (Beckman Avanti J-25I) Spectramax M5 (Molecular Devices) BugBuster 10X protein extraction reagent (Novagen) Protease inhibitor cocktail (Roche) 2X Binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM Imidazole, and 0.04% NaN3 ) 2X GST binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl and 0.04% NaN3 ) Benzonase (Novagen) 50 mM Phenylmethylsulfonyl fluoride (PMSF) Dithiothreitol (DTT) (Sigma) Chromatography column (10x100 mm) (Sigma) Column stand with clamp Ni-NTA resin (Qiagen) Glutathione agarose resin (Amersham Biosciences) Reduced glutathione (Sigma) Wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM Imidazole) Elution buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM Imidazole) 1X GST wash buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) GST elution buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM Glutathione reduced) Dialysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT) Phosphate buffered saline (PBS) 15 ml 10kD and 30kD Centricon (Millipore) Cold room 2X SDS sample buffer with DTT 4–12% Bis-Tris gradient gel (Invitrogen) NuPAGE MOPS SDS running buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) SimplyBlue Stain (Invitrogen) 1.5 ml Sterile microcentrifuge tubes Bradford reagent (Bio-rad) Bradford reagent (Bio-rad) .. 1 Culture a single colony of freshly transformed cells in 25 ml of LB broth containing 0.5 mg/ml kanamycin and 0.05 mg/ml chloroamphenicol at 225 rpm for 12 hrs at 37 ºC. (For E1: LB broth contains 0.5 mg/ml kanamycin, 0.05 mg/ml chloroamphenicol and 0.1 mg/ml ampicillin).

    Blocking Assay:

    Article Title: Assays for investigating deSUMOylation enzymes (ms# CP-11-0260)
    Article Snippet: .. YSE 10X reaction buffer Milli-Q-purified water Human SENP catalytic domain (320 nM stock ) SENP buffer 3X sample buffer Heat block or bath at 37°C 0.5 mL microcentrifuge tubes Ice bucket with ice 4–12% Bis-Tris gel (Life technologies) NuPAGE MOPS running SDS buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) .. Equilibrate heating block or water bath to 37 °C.

    Article Title: Assays for investigating deSUMOylation enzymes (ms# CP-11-0260)
    Article Snippet: .. HeLa cells (ATCC, maintained with Complete DMEM) DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen, Carlsbad CA) Penicillin, streptomycin (Invitrogen, Carlsbad CA) Fetal bovine serum (FBS) (Invitrogen, Carlsbad CA) Phosphate Buffered Saline (PBS) Lysis buffer (at 4 °C) 3X Sample buffer Blocking buffer ( Odyessey, Licor) PBST Anti-SENP1 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP2 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP3 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP5 mouse monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-HA mouse monoclonal primary antibodies (Sigma, Missouri) Anti-mouse secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) Anti-rabbit secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) HA-SUMO1-VS (Boston Biochem, Cambridge MA) HA-SUMO2-VS (Boston Biochem, Cambridge MA) 100 mm Tissue culture plates (Corning, Corning NY) 1.5 ml pop-top microcentrifugetubes (Denville) Cell scraper (BD Biosciences ) Ice bucket with ice Tabletop microcentrifuge at 4 °C (Eppendorf, model No. 5417R) Sonicator XL with a micro tip probe (Misonix, farmingdale, NY) Boiling water bath 10-well 4–12% gradient Bis-Tris gel (Life technologies) NuPAGE MOPS running SDS buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) Trans blot turbo transfer system apparatus (Bio-rad) 0.2 μM PVDF trans blot turbo membrane (Bio-rad) Super signal west Pico chemiluminescent substrate (Thermo scientific) Blue X-ray Film (Bioland) .. Incubate 50 μl of cell lysate, which contains SENPs, or recombinant SENPs (100 nM) with 1-5 μM of 1VS or 2VS for 15 min at 25 °C.

    Immunoprecipitation:

    Article Title: Meiotic cohesins mediate initial loading of HORMAD1 to the chromosomes and coordinate SC formation during meiotic prophase
    Article Snippet: .. The immunoprecipitated proteins were run on 4–12% Bis-Tris NuPAGE (NP0322, Thermo Fisher Scientific) in MOPS-SDS buffer or 7% Tris-Acetate NuPAGE (EA030552, Thermo Fisher Scientific) in Tris-Acetate-SDS buffer for immunoblotting or LC-MS/MS analysis. .. Immunoblots were detected by VeritBlot for IP detection reagent (HRP) (ab131366, abcam, 1:3000 dilution).

    Protein Extraction:

    Article Title: Biochemical Analysis of Protein SUMOylation
    Article Snippet: .. Luria-Bertani (LB) broth (Sigma), (20g LB powder/1 L H2 O), autoclaved 500 mg/ml Kanamycin stock solution (Fisher Scientific), 0.22 um filter-sterilized 100 mg/ml Ampicillin stock solution (Fisher Scientific), 0.22 um filter-sterilized 50 mg/ml Chloroamphenicol stock solution (Fisher Scientific) in ethanol, 0.22 um filter-sterilized 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientifics) Milli-Q-water Shaker Incubator (Thermo-Electron Corp.) Ultracentrifuge (Beckman Avanti J-25I) Spectramax M5 (Molecular Devices) BugBuster 10X protein extraction reagent (Novagen) Protease inhibitor cocktail (Roche) 2X Binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM Imidazole, and 0.04% NaN3 ) 2X GST binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl and 0.04% NaN3 ) Benzonase (Novagen) 50 mM Phenylmethylsulfonyl fluoride (PMSF) Dithiothreitol (DTT) (Sigma) Chromatography column (10x100 mm) (Sigma) Column stand with clamp Ni-NTA resin (Qiagen) Glutathione agarose resin (Amersham Biosciences) Reduced glutathione (Sigma) Wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM Imidazole) Elution buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM Imidazole) 1X GST wash buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) GST elution buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM Glutathione reduced) Dialysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT) Phosphate buffered saline (PBS) 15 ml 10kD and 30kD Centricon (Millipore) Cold room 2X SDS sample buffer with DTT 4–12% Bis-Tris gradient gel (Invitrogen) NuPAGE MOPS SDS running buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) SimplyBlue Stain (Invitrogen) 1.5 ml Sterile microcentrifuge tubes Bradford reagent (Bio-rad) Bradford reagent (Bio-rad) .. 1 Culture a single colony of freshly transformed cells in 25 ml of LB broth containing 0.5 mg/ml kanamycin and 0.05 mg/ml chloroamphenicol at 225 rpm for 12 hrs at 37 ºC. (For E1: LB broth contains 0.5 mg/ml kanamycin, 0.05 mg/ml chloroamphenicol and 0.1 mg/ml ampicillin).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Two p53 tetramers bind one consensus DNA response element
    Article Snippet: .. 1x MOPS SDS buffer (Invitrogen) was used for the denaturing PAGE. .. Anode (25 mM imidazole. pH 7) and cathode (50 mM tricine, 7.5mM imidazole, 0.002% Coomassie blue-G250. pH 7) buffers were used for the Native PAGE.

    Staining:

    Article Title: Biochemical Analysis of Protein SUMOylation
    Article Snippet: .. Luria-Bertani (LB) broth (Sigma), (20g LB powder/1 L H2 O), autoclaved 500 mg/ml Kanamycin stock solution (Fisher Scientific), 0.22 um filter-sterilized 100 mg/ml Ampicillin stock solution (Fisher Scientific), 0.22 um filter-sterilized 50 mg/ml Chloroamphenicol stock solution (Fisher Scientific) in ethanol, 0.22 um filter-sterilized 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientifics) Milli-Q-water Shaker Incubator (Thermo-Electron Corp.) Ultracentrifuge (Beckman Avanti J-25I) Spectramax M5 (Molecular Devices) BugBuster 10X protein extraction reagent (Novagen) Protease inhibitor cocktail (Roche) 2X Binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM Imidazole, and 0.04% NaN3 ) 2X GST binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl and 0.04% NaN3 ) Benzonase (Novagen) 50 mM Phenylmethylsulfonyl fluoride (PMSF) Dithiothreitol (DTT) (Sigma) Chromatography column (10x100 mm) (Sigma) Column stand with clamp Ni-NTA resin (Qiagen) Glutathione agarose resin (Amersham Biosciences) Reduced glutathione (Sigma) Wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM Imidazole) Elution buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM Imidazole) 1X GST wash buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) GST elution buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM Glutathione reduced) Dialysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT) Phosphate buffered saline (PBS) 15 ml 10kD and 30kD Centricon (Millipore) Cold room 2X SDS sample buffer with DTT 4–12% Bis-Tris gradient gel (Invitrogen) NuPAGE MOPS SDS running buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) SimplyBlue Stain (Invitrogen) 1.5 ml Sterile microcentrifuge tubes Bradford reagent (Bio-rad) Bradford reagent (Bio-rad) .. 1 Culture a single colony of freshly transformed cells in 25 ml of LB broth containing 0.5 mg/ml kanamycin and 0.05 mg/ml chloroamphenicol at 225 rpm for 12 hrs at 37 ºC. (For E1: LB broth contains 0.5 mg/ml kanamycin, 0.05 mg/ml chloroamphenicol and 0.1 mg/ml ampicillin).

    Modification:

    Article Title: Assays for investigating deSUMOylation enzymes (ms# CP-11-0260)
    Article Snippet: .. HeLa cells (ATCC, maintained with Complete DMEM) DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen, Carlsbad CA) Penicillin, streptomycin (Invitrogen, Carlsbad CA) Fetal bovine serum (FBS) (Invitrogen, Carlsbad CA) Phosphate Buffered Saline (PBS) Lysis buffer (at 4 °C) 3X Sample buffer Blocking buffer ( Odyessey, Licor) PBST Anti-SENP1 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP2 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP3 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP5 mouse monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-HA mouse monoclonal primary antibodies (Sigma, Missouri) Anti-mouse secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) Anti-rabbit secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) HA-SUMO1-VS (Boston Biochem, Cambridge MA) HA-SUMO2-VS (Boston Biochem, Cambridge MA) 100 mm Tissue culture plates (Corning, Corning NY) 1.5 ml pop-top microcentrifugetubes (Denville) Cell scraper (BD Biosciences ) Ice bucket with ice Tabletop microcentrifuge at 4 °C (Eppendorf, model No. 5417R) Sonicator XL with a micro tip probe (Misonix, farmingdale, NY) Boiling water bath 10-well 4–12% gradient Bis-Tris gel (Life technologies) NuPAGE MOPS running SDS buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) Trans blot turbo transfer system apparatus (Bio-rad) 0.2 μM PVDF trans blot turbo membrane (Bio-rad) Super signal west Pico chemiluminescent substrate (Thermo scientific) Blue X-ray Film (Bioland) .. Incubate 50 μl of cell lysate, which contains SENPs, or recombinant SENPs (100 nM) with 1-5 μM of 1VS or 2VS for 15 min at 25 °C.

    Lysis:

    Article Title: Assays for investigating deSUMOylation enzymes (ms# CP-11-0260)
    Article Snippet: .. HeLa cells (ATCC, maintained with Complete DMEM) DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen, Carlsbad CA) Penicillin, streptomycin (Invitrogen, Carlsbad CA) Fetal bovine serum (FBS) (Invitrogen, Carlsbad CA) Phosphate Buffered Saline (PBS) Lysis buffer (at 4 °C) 3X Sample buffer Blocking buffer ( Odyessey, Licor) PBST Anti-SENP1 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP2 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP3 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP5 mouse monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-HA mouse monoclonal primary antibodies (Sigma, Missouri) Anti-mouse secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) Anti-rabbit secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) HA-SUMO1-VS (Boston Biochem, Cambridge MA) HA-SUMO2-VS (Boston Biochem, Cambridge MA) 100 mm Tissue culture plates (Corning, Corning NY) 1.5 ml pop-top microcentrifugetubes (Denville) Cell scraper (BD Biosciences ) Ice bucket with ice Tabletop microcentrifuge at 4 °C (Eppendorf, model No. 5417R) Sonicator XL with a micro tip probe (Misonix, farmingdale, NY) Boiling water bath 10-well 4–12% gradient Bis-Tris gel (Life technologies) NuPAGE MOPS running SDS buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) Trans blot turbo transfer system apparatus (Bio-rad) 0.2 μM PVDF trans blot turbo membrane (Bio-rad) Super signal west Pico chemiluminescent substrate (Thermo scientific) Blue X-ray Film (Bioland) .. Incubate 50 μl of cell lysate, which contains SENPs, or recombinant SENPs (100 nM) with 1-5 μM of 1VS or 2VS for 15 min at 25 °C.

    Binding Assay:

    Article Title: Biochemical Analysis of Protein SUMOylation
    Article Snippet: .. Luria-Bertani (LB) broth (Sigma), (20g LB powder/1 L H2 O), autoclaved 500 mg/ml Kanamycin stock solution (Fisher Scientific), 0.22 um filter-sterilized 100 mg/ml Ampicillin stock solution (Fisher Scientific), 0.22 um filter-sterilized 50 mg/ml Chloroamphenicol stock solution (Fisher Scientific) in ethanol, 0.22 um filter-sterilized 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientifics) Milli-Q-water Shaker Incubator (Thermo-Electron Corp.) Ultracentrifuge (Beckman Avanti J-25I) Spectramax M5 (Molecular Devices) BugBuster 10X protein extraction reagent (Novagen) Protease inhibitor cocktail (Roche) 2X Binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM Imidazole, and 0.04% NaN3 ) 2X GST binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl and 0.04% NaN3 ) Benzonase (Novagen) 50 mM Phenylmethylsulfonyl fluoride (PMSF) Dithiothreitol (DTT) (Sigma) Chromatography column (10x100 mm) (Sigma) Column stand with clamp Ni-NTA resin (Qiagen) Glutathione agarose resin (Amersham Biosciences) Reduced glutathione (Sigma) Wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM Imidazole) Elution buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM Imidazole) 1X GST wash buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) GST elution buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM Glutathione reduced) Dialysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT) Phosphate buffered saline (PBS) 15 ml 10kD and 30kD Centricon (Millipore) Cold room 2X SDS sample buffer with DTT 4–12% Bis-Tris gradient gel (Invitrogen) NuPAGE MOPS SDS running buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) SimplyBlue Stain (Invitrogen) 1.5 ml Sterile microcentrifuge tubes Bradford reagent (Bio-rad) Bradford reagent (Bio-rad) .. 1 Culture a single colony of freshly transformed cells in 25 ml of LB broth containing 0.5 mg/ml kanamycin and 0.05 mg/ml chloroamphenicol at 225 rpm for 12 hrs at 37 ºC. (For E1: LB broth contains 0.5 mg/ml kanamycin, 0.05 mg/ml chloroamphenicol and 0.1 mg/ml ampicillin).

    SDS Page:

    Article Title: Biochemical Analysis of Protein SUMOylation
    Article Snippet: .. Luria-Bertani (LB) broth (Sigma), (20g LB powder/1 L H2 O), autoclaved 500 mg/ml Kanamycin stock solution (Fisher Scientific), 0.22 um filter-sterilized 100 mg/ml Ampicillin stock solution (Fisher Scientific), 0.22 um filter-sterilized 50 mg/ml Chloroamphenicol stock solution (Fisher Scientific) in ethanol, 0.22 um filter-sterilized 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientifics) Milli-Q-water Shaker Incubator (Thermo-Electron Corp.) Ultracentrifuge (Beckman Avanti J-25I) Spectramax M5 (Molecular Devices) BugBuster 10X protein extraction reagent (Novagen) Protease inhibitor cocktail (Roche) 2X Binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM Imidazole, and 0.04% NaN3 ) 2X GST binding buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl and 0.04% NaN3 ) Benzonase (Novagen) 50 mM Phenylmethylsulfonyl fluoride (PMSF) Dithiothreitol (DTT) (Sigma) Chromatography column (10x100 mm) (Sigma) Column stand with clamp Ni-NTA resin (Qiagen) Glutathione agarose resin (Amersham Biosciences) Reduced glutathione (Sigma) Wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM Imidazole) Elution buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 300 mM Imidazole) 1X GST wash buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT) GST elution buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM Glutathione reduced) Dialysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM DTT) Phosphate buffered saline (PBS) 15 ml 10kD and 30kD Centricon (Millipore) Cold room 2X SDS sample buffer with DTT 4–12% Bis-Tris gradient gel (Invitrogen) NuPAGE MOPS SDS running buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) SimplyBlue Stain (Invitrogen) 1.5 ml Sterile microcentrifuge tubes Bradford reagent (Bio-rad) Bradford reagent (Bio-rad) .. 1 Culture a single colony of freshly transformed cells in 25 ml of LB broth containing 0.5 mg/ml kanamycin and 0.05 mg/ml chloroamphenicol at 225 rpm for 12 hrs at 37 ºC. (For E1: LB broth contains 0.5 mg/ml kanamycin, 0.05 mg/ml chloroamphenicol and 0.1 mg/ml ampicillin).

    Article Title: Assays for investigating deSUMOylation enzymes (ms# CP-11-0260)
    Article Snippet: .. YSE 10X reaction buffer Milli-Q-purified water Human SENP catalytic domain (320 nM stock ) SENP buffer 3X sample buffer Heat block or bath at 37°C 0.5 mL microcentrifuge tubes Ice bucket with ice 4–12% Bis-Tris gel (Life technologies) NuPAGE MOPS running SDS buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) .. Equilibrate heating block or water bath to 37 °C.

    Article Title: Assays for investigating deSUMOylation enzymes (ms# CP-11-0260)
    Article Snippet: .. HeLa cells (ATCC, maintained with Complete DMEM) DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen, Carlsbad CA) Penicillin, streptomycin (Invitrogen, Carlsbad CA) Fetal bovine serum (FBS) (Invitrogen, Carlsbad CA) Phosphate Buffered Saline (PBS) Lysis buffer (at 4 °C) 3X Sample buffer Blocking buffer ( Odyessey, Licor) PBST Anti-SENP1 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP2 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP3 rabbit monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-SENP5 mouse monoclonal primary antibodies (Epitomics, Burlingame CA) Anti-HA mouse monoclonal primary antibodies (Sigma, Missouri) Anti-mouse secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) Anti-rabbit secondary antibodies (IgG (H+L) HRP-conjugated) (Promega) HA-SUMO1-VS (Boston Biochem, Cambridge MA) HA-SUMO2-VS (Boston Biochem, Cambridge MA) 100 mm Tissue culture plates (Corning, Corning NY) 1.5 ml pop-top microcentrifugetubes (Denville) Cell scraper (BD Biosciences ) Ice bucket with ice Tabletop microcentrifuge at 4 °C (Eppendorf, model No. 5417R) Sonicator XL with a micro tip probe (Misonix, farmingdale, NY) Boiling water bath 10-well 4–12% gradient Bis-Tris gel (Life technologies) NuPAGE MOPS running SDS buffer (Invitrogen) SDS-PAGE apparatus (Invitrogen) Trans blot turbo transfer system apparatus (Bio-rad) 0.2 μM PVDF trans blot turbo membrane (Bio-rad) Super signal west Pico chemiluminescent substrate (Thermo scientific) Blue X-ray Film (Bioland) .. Incubate 50 μl of cell lysate, which contains SENPs, or recombinant SENPs (100 nM) with 1-5 μM of 1VS or 2VS for 15 min at 25 °C.

    Article Title: Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells . Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells
    Article Snippet: .. Protein samples were mixed with NuPAGE sample buffer containing dithiothreitol (DTT) and proceeded for SDS‐PAGE gel electrophoresis (NuPAGE precast 10% or 4–12% Bis‐tris gels in NuPAGE MOPS SDS running buffer, Invitrogen, Waltham, MA) in NOVEX chambers under reducing/denaturing conditions. .. To indicate the molecular weight of proteins we used a benchmark protein ladder (Invitrogen).

    Article Title: Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41 *
    Article Snippet: .. The viral pellet was then resuspended in MOPS and NuPAGE SDS-PAGE buffer, heated for 10 min at 70 °C, and loaded onto NuPAGE 4–12% bis-tris gel (Invitrogen). .. Gels were electrophoresed for 50 min at 200 volts followed by transfer on PVDF membrane using the iBlot system (Invitrogen).

    Article Title: Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation
    Article Snippet: .. Once cooled to room temperature, 10 μl of pH 8 buffer (100 m m sodium phosphate) was added to neutralize followed by 6 μl of 1 m m maleimide-conjugated PEG, M r ∼5000 (MilliporeSigma), and 4 μl of 4× NuPAGE SDS-PAGE gel-loading buffer (Thermo Fisher). .. The reaction mixtures were incubated at 50 °C for 2 h and then analyzed directly by SDS-PAGE with Coomassie stain (Thermo Fisher).

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    Thermo Fisher nupage sds page gel loading buffer
    Evidence of disulfide transfer among soluble HγD molecules. An <t>SDS-PAGE</t> gel of samples incubated with maleimide-PEG to quantify the distribution of the number of free thiol groups per protein molecule is shown. When reduced WT protein was mixed with CCC(ox.) and incubated for 1 h at pH 5, at which disulfide exchange is inhibited ( left lane ), the resulting molecular population at the end of the reaction consisted largely of 6- and 1-free-thiol populations, corresponding to the starting samples. When pH in the same mixture was set to 8, allowing disulfide exchange ( right lane ), after 1 h the population shifted toward 4- and 3-free-thiol positions, corresponding to oxidized WT and reduced C18T/C41A/C78A, respectively.
    Nupage Sds Page Gel Loading Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Evidence of disulfide transfer among soluble HγD molecules. An SDS-PAGE gel of samples incubated with maleimide-PEG to quantify the distribution of the number of free thiol groups per protein molecule is shown. When reduced WT protein was mixed with CCC(ox.) and incubated for 1 h at pH 5, at which disulfide exchange is inhibited ( left lane ), the resulting molecular population at the end of the reaction consisted largely of 6- and 1-free-thiol populations, corresponding to the starting samples. When pH in the same mixture was set to 8, allowing disulfide exchange ( right lane ), after 1 h the population shifted toward 4- and 3-free-thiol positions, corresponding to oxidized WT and reduced C18T/C41A/C78A, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation

    doi: 10.1074/jbc.RA118.004551

    Figure Lengend Snippet: Evidence of disulfide transfer among soluble HγD molecules. An SDS-PAGE gel of samples incubated with maleimide-PEG to quantify the distribution of the number of free thiol groups per protein molecule is shown. When reduced WT protein was mixed with CCC(ox.) and incubated for 1 h at pH 5, at which disulfide exchange is inhibited ( left lane ), the resulting molecular population at the end of the reaction consisted largely of 6- and 1-free-thiol populations, corresponding to the starting samples. When pH in the same mixture was set to 8, allowing disulfide exchange ( right lane ), after 1 h the population shifted toward 4- and 3-free-thiol positions, corresponding to oxidized WT and reduced C18T/C41A/C78A, respectively.

    Article Snippet: Once cooled to room temperature, 10 μl of pH 8 buffer (100 m m sodium phosphate) was added to neutralize followed by 6 μl of 1 m m maleimide-conjugated PEG, M r ∼5000 (MilliporeSigma), and 4 μl of 4× NuPAGE SDS-PAGE gel-loading buffer (Thermo Fisher).

    Techniques: SDS Page, Incubation, Countercurrent Chromatography

    Relative abundance of the 10 most abundant glycopeptides of prostate-specific antigen after either capturing (from spiked female urine pool) or no capturing (PSA standard, 1.5 μg) procedure. All samples were loaded on to a SDS-PAGE gel, the approximately ∼32 kDa band was excised from the gel and underwent a tryptic in-gel digestion prior to CE-ESI-MS analysis. Error bars represent the standard deviation ( N = 3). PEP illustrates the tryptic peptide sequence N 69 K.

    Journal: Analytical Chemistry

    Article Title: An In-Depth Glycosylation Assay for Urinary Prostate-Specific Antigen

    doi: 10.1021/acs.analchem.7b04281

    Figure Lengend Snippet: Relative abundance of the 10 most abundant glycopeptides of prostate-specific antigen after either capturing (from spiked female urine pool) or no capturing (PSA standard, 1.5 μg) procedure. All samples were loaded on to a SDS-PAGE gel, the approximately ∼32 kDa band was excised from the gel and underwent a tryptic in-gel digestion prior to CE-ESI-MS analysis. Error bars represent the standard deviation ( N = 3). PEP illustrates the tryptic peptide sequence N 69 K.

    Article Snippet: Immobilization of the nanobodies was confirmed by analyzing the supernatant on a NuPAGE SDS-PAGE gel (Thermo Fisher, Waltham, MA) with NuPAGE MOPS SDS running buffer (Thermo Fisher) and after staining with Coomassie G-250 (SimplyBlue SafeStain, Colloidal Blue Staining Kit, Thermo Fisher).

    Techniques: SDS Page, Mass Spectrometry, Standard Deviation, Sequencing

    HORMAD1 interacts with cohesin and chromosome axis proteins. (A) WT neonatal male mice were subjected to consecutive injection of a RA synthesis inhibitor WIN18,446, followed by injection of RA. Seminiferous tubule section at 8 days post treatment were stained for SYCP3, STRA8, SYCP1(Cy5) and DAPI. Scale bar: 25 μm. Proportion of the meiotic prophase stages of spermatocytes is shown on the right. n: number of SYCP3 positive cells examined. preL: SYCP3+/STRA8+ preleptotene, Lep: SYCP3+/STRA8- leptotene. As weak staining of SYCP1 over spermatocyte nuclei was detected even in preleptotene and leptotene, spermatocytes with thread-like pattern of SYCP1 staining was defined as Z./P.: zygotene or pachytene. (B) The immunoprecipitates by anti-HORMAD1 antibody from the chromatin-bound fraction of the testis were subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analyses. The proteins identified by the LC-MS/MS analyses are listed with the number of peptide hits and Mascot scores. (C) The immunoprecipitates by anti-REC8 and anti-RAD21L antibodies from the chromatin-bound fraction of the testis were subjected to LC-MS/MS analyses as in (B). (D) WB analysis of HORMAD1 immunoprecipitates with indicated antibodies. HORMAD1 immunoprecipitates were run on 4–12% NuPAGE Bis-Tris in MOPS-SDS buffer. * indicates a band that cross-reacted to IgG heavy chain. (E) WB analysis of REC8 and RAD21L immunoprecipitates with indicated antibodies. REC8 and RAD21L immunoprecipitates were run on 4–12% NuPAGE Bis-Tris in MOPS-SDS buffer (for immunoblots of REC8, HORMAD1 and Actin) and 7% NuPAGE Tris-Acetate in Tris-Acetate-SDS buffer (for immunoblots of SMC3, STAG3 and RAD21L). * indicates a band that cross-reacted to IgG heavy chain.

    Journal: PLoS Genetics

    Article Title: Meiotic cohesins mediate initial loading of HORMAD1 to the chromosomes and coordinate SC formation during meiotic prophase

    doi: 10.1371/journal.pgen.1009048

    Figure Lengend Snippet: HORMAD1 interacts with cohesin and chromosome axis proteins. (A) WT neonatal male mice were subjected to consecutive injection of a RA synthesis inhibitor WIN18,446, followed by injection of RA. Seminiferous tubule section at 8 days post treatment were stained for SYCP3, STRA8, SYCP1(Cy5) and DAPI. Scale bar: 25 μm. Proportion of the meiotic prophase stages of spermatocytes is shown on the right. n: number of SYCP3 positive cells examined. preL: SYCP3+/STRA8+ preleptotene, Lep: SYCP3+/STRA8- leptotene. As weak staining of SYCP1 over spermatocyte nuclei was detected even in preleptotene and leptotene, spermatocytes with thread-like pattern of SYCP1 staining was defined as Z./P.: zygotene or pachytene. (B) The immunoprecipitates by anti-HORMAD1 antibody from the chromatin-bound fraction of the testis were subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analyses. The proteins identified by the LC-MS/MS analyses are listed with the number of peptide hits and Mascot scores. (C) The immunoprecipitates by anti-REC8 and anti-RAD21L antibodies from the chromatin-bound fraction of the testis were subjected to LC-MS/MS analyses as in (B). (D) WB analysis of HORMAD1 immunoprecipitates with indicated antibodies. HORMAD1 immunoprecipitates were run on 4–12% NuPAGE Bis-Tris in MOPS-SDS buffer. * indicates a band that cross-reacted to IgG heavy chain. (E) WB analysis of REC8 and RAD21L immunoprecipitates with indicated antibodies. REC8 and RAD21L immunoprecipitates were run on 4–12% NuPAGE Bis-Tris in MOPS-SDS buffer (for immunoblots of REC8, HORMAD1 and Actin) and 7% NuPAGE Tris-Acetate in Tris-Acetate-SDS buffer (for immunoblots of SMC3, STAG3 and RAD21L). * indicates a band that cross-reacted to IgG heavy chain.

    Article Snippet: The immunoprecipitated proteins were run on 4–12% Bis-Tris NuPAGE (NP0322, Thermo Fisher Scientific) in MOPS-SDS buffer or 7% Tris-Acetate NuPAGE (EA030552, Thermo Fisher Scientific) in Tris-Acetate-SDS buffer for immunoblotting or LC-MS/MS analysis.

    Techniques: Mouse Assay, Injection, Staining, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot

    ( a ) The trace from a typical size-exclusion run of purified base is shown. The peak between the void and the assembled base contains misassembled base and on a gel looks similar to properly assembled base. ( b ) Gel samples from a typical base purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

    doi: 10.1007/978-1-4939-8706-1_15

    Figure Lengend Snippet: ( a ) The trace from a typical size-exclusion run of purified base is shown. The peak between the void and the assembled base contains misassembled base and on a gel looks similar to properly assembled base. ( b ) Gel samples from a typical base purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Article Snippet: 20 × NuPAGE MOPS running buffer (Thermo Fisher).

    Techniques: Purification, Staining

    ( a ) The trace from a typical size-exclusion run of purified lid is shown. The peak after the assembled lid contains mostly cleaved MBP. ( b ) Gel samples from a typical lid purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

    doi: 10.1007/978-1-4939-8706-1_15

    Figure Lengend Snippet: ( a ) The trace from a typical size-exclusion run of purified lid is shown. The peak after the assembled lid contains mostly cleaved MBP. ( b ) Gel samples from a typical lid purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Article Snippet: 20 × NuPAGE MOPS running buffer (Thermo Fisher).

    Techniques: Purification, Staining