Kidney and cell line samples were resuspended by sonication in a 1:100 w/v ratio in Tris HCl, 25 mM, pH 7.4 buffer in the presence of proteinase inhibitors, boiled in final sample buffer (2% β-mecaptoethanol, 2% SDS), then centrifuged for 5 minutes at 14,000 rpm. The solubilized proteins were separated by electrophoresis on a 4–20% gradient SDS-PAGE gel in MOPS buffer (GenScript #M00656), and then blotted on a nitrocellulose membrane. The PageRuler (Thermo #26616) was used for size reference. The membrane was blocked in PBS supplemented with non-fat dry milk (5%) and Tween-20 (0.5%) for one hour at room temperature, and then probed with primary antibodies (ATF4 Cell Signaling, D4B8, #11815, 1:1,000 dilution, Asns, Proteintech. #14681–1-AP, lot-00048645, 1:1,000 dilution; Ndufa4l2, Thermo, #PA5–39268, lot-UJ2859452, 1:1,000 dilution; β-actin, Millipore #MAB1501, lot-2951837, 1:5,000 dilution) overnight at 4°C. The staining was detected using anti-rabbit secondary antibody (Jackson Immuno-Research, # 715-035-152, lot-121111) or anti-mouse secondary antibody (Jackson Immuno-Research, # 715-035-150, lot-120917) in a 1:10,000 dilution, followed by ECL detection (Pierce, # 32109).
Mops buffer
Manufactured by GenScript
Sourced in United States
MOPS buffer is a chemical solution commonly used in molecular biology and biochemistry applications. It serves as a buffering agent to maintain a stable pH environment during various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (1 mentions)
Anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Pageruler, by Thermo Fisher Scientific (1 mentions)
Anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Uvp chemstudio, by Analytik Jena (1 mentions)
Phosphostop, by Roche (1 mentions)
Expressplustm page gel, by GenScript (1 mentions)
4 protocols using mops buffer
1
Quantitative Protein Expression Analysis
2
Protein Expression Analysis of Neural Crest Cells
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Whole cell lysates were obtained from day 12 NCCs or day 20 SNs. Cells were washed once with PBS and incubated with RIPA buffer (ThermoFisher) with 1 mM PMSF and Phospho-STOP (Roche) for 15min on ice. Cells were then vortexed for 10 s and centrifuged at 12,000 RPM for 10 min at 4°C. Protein concentration from supernatants was measured and ran in 7.5% polyacrylamide gels using MOPS buffer (GenScript) at 130 V. Proteins were transferred to a nitrocellulose membrane and blocked for 30 min in 5% non-fat dry milk in 0.1% Tween-20 in TBS (TBS-T, 50 mM Tris-HCl, 150 mM NaCl, pH7.6). Primary antibodies were added to blocking buffer (SOX10 – 1:1000, BRN3A – 1:1000, and Actin – 1:5000) and membranes were incubated overnight at 4°C. Blots were then washed 3X with 0.1% TBS-T and incubated with goat anti-mouse HRP antibody (1:5000) for 1 h at room temperature. Blots were washed 3X with 0.1% TBS-T followed by incubation with Clarity Western ECL Substrate (BioRad). Chemiluminescence signal was detected using UVP ChemStudio (Analytic Jena).
3
Recombinant Pr77Gag Protein Purification
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SDS-PAGE and western blotting were used to monitor the expression and purification of recombinant Pr77Gag-His6-tag protein. Protein samples were analyzed on 4–12% ExpressPlusTM PAGE gel (GenScript, Piscataway, NJ, USA), electrophoresed under reducing conditions using 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (GenScript), and stained with Coomassie Brilliant Blue. For western blot analyses, duplicate gels were transferred onto nitrocellulose membranes and probed with α-MMTV p27 CA monoclonal antibody Blue 7 [68 (link)] and an α-His6 monoclonal antibody-horseradish peroxidase (HRP) conjugate (Sigma-Aldrich).
4
Recombinant Pr78Gag Protein Expression
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Expression and purification of recombinant Pr78Gag-His6-tag protein was monitored by SDS-PAGE and western blotting. Briefly, protein samples were mixed with 6X SDS dye, boiled for 5 minutes before loading onto a 4–12% ExpressPlusTM PAGE gel (GenScript), electrophoresed under reducing conditions using MOPS buffer (GenScript), followed by their staining with Coomassie Brilliant Blue. Recombinant Pr78Gag expression and purification was further monitored by transferring non-stained gels onto a nitrocellulose membrane and blotting with anti-rabbit MPMV Gag/Pol Pr78 polyserum (kindly provided by Dr. Eric Hunter, Emory University, Atlanta, GA) and with an anti-His6 monoclonal antibody-HRP conjugate (Sigma-Aldrich).
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