Cell lysates were prepared with RIPA. Protein in the supernatant was extracted, and its concentration was measured using the BCA Protein Assay Kit. An equal amount (50 ug) of total cellular protein was electrophoresed by denaturing 12.5% SDS-PAGE and transferred to 0.22um polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The locations of proteins of interest were detected by primary antibodies for overnight at 4°C. ERp19 antibody was from Abcam;GAPDH antibody was from Kangchen Bio-tech; FAK, FAK pY397, paxillin, paxillin pY118, ERK1/2 and p-ERK1/2 antibodies were from Cell Signaling Biotechnology. After HRP conjugated-secondary antibody bound to the primary antibodies, the proteins were visualized using enhanced chemiluminescence (ECL) reagent.
Gapdh antibody
Manufactured by Kangchen Biotech
Sourced in United States
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) antibody is a primary antibody used in various laboratory techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry. The antibody specifically binds to the GAPDH protein, which is a key enzyme involved in the glycolytic pathway.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Py397 fak, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Paxillin, by Cell Signaling Technology (1 mentions)
Paxillin py118, by Cell Signaling Technology (1 mentions)
Flag antibody, by Merck Group (1 mentions)
Bmp 6 antibody, by Santa Cruz Biotechnology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Antibody against ubiquitin 6c1.17, by BD (1 mentions)
5 protocols using gapdh antibody
1
Western Blot Analysis of Cellular Proteins
2
Immunoblotting Antibody Protocol
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Immunoblotting experiments were performed according to standard procedures. The following antibodies were used: BMP-6 antibody (1∶50; Santa Cruz Biotechnology, Santa Cruz, CA), FLAG antibody (1∶2,000; Sigma-Aldrich St. Louis, MO), and GAPDH antibody [used as the internal control (1∶10,000; Kang-Chen Bio-tech Shanghai, China)].
3
Western Blot Analysis of Paxillin
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Cells were lysed using RIPA cell lysis buffer (Kangwei) supplemented with protease inhibitor cocktail (Cell Signaling Biotechnology). The amount of total protein was quantified using a protein assay kit (Bio-Rad). Protein samples were loaded onto 12.5% SDS-PAGE gels and then transferred onto PVDF membranes. The membranes were blocked in TBS-T buffer containing 5% non-fat dry milk and hybridized with a primary antibody. Paxillin (Tyr118) antibody was from Abcam, GAPDH antibody was from Kangchen Bio-tech, and all other primary and secondary antibodies were from Cell Signaling Biotechnology. Finally, membranes were incubated with HRP-conjugated secondary antibody. Protein bands were visualized using ECL reagent (Thermo) on a Tanon detection system.
4
Western Blot Analysis of Cell Signaling
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Briefly, BGC823 cells were dealt with different drugs for 36h and then lysed with lysis bufer (Beyotime Institute of Biotechnology, Shanghai, China). All the selected protein extracts were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to a polyvinylidene difluoridex (PVDF) membrane (Beyotime Institute of Biotechnology). The membranes were blocked with 5% fresh nonfat milk dissolved in tris buffer solution tween (TBST) and incubated with primary and secondary antibodies, respectively. Signals were visualized using an ECL kit (Bio-Rad, Hercules, CA). The bands were semiquantified using Image J software. AKT, p-AKT, ERK, p-ERK, cyclinD1, p21 and p27 antibodies were purchased from Cell Signaling Technology (MA, USA), and the GAPDH antibody was from Kangchen Bio-tech (Shanghai, China).
5
Plasmid Construction and Antibody Characterization
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WDR79 or UHRF1, the full‐length mRNA sequences, were PCR‐amplified from human cDNA and subcloned into pCMV‐Tag2B to create Flag‐tagged WDR79 expression plasmids. The WDR79 and UHRF1 antibodies were from Bethyl Laboratories (Montgomer y, TX, USA), the GAPDH antibody was obtained from KangChen Bio‐tech Inc (Shanghai, China) and the antibody against ubiquitin (6C1.17) was from BD Pharmingen (Franklin Lakes, NJ, USA).
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