Western blotting GAPDH

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

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3 years ago

3 years ago by Natalia Penaranda

Best Antibody

which is a good monoclonal anibody for this protein?

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Found 3 matching solutions for this experiment

GAPDH Loading Control Monoclonal Antibody (GA1R)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	For detection of transiently expressed proteins, a total of 2.5 μg DNA, including pHT1-3 and/or pTat and/or empty pEF.Bos vector (see above), was transfected using Lipofectamine 3000 (Invitrogen) in 8.0E+05 293T cells. After 48 hrs, the cells were washed using PBS, and lysed using RIPA buffer containing 150 mM KCl and a protease inhibitor mixture (Thermo Fisher Scientific). Lysates were incubated for 10 min at 95°C in 2X Laemmli buffer (BioRad) supplemented with 5% DTT, and used for SDS-PAGE analysis in a 15% resolving gel. The proteins were then transferred to a PVDF membrane (BioRad), which was blocked in 5% milk in TBS and probed with specific primary Abs, which include anti-Myc (ab32 from mouse and ab9106 from rabbit, Abcam), anti-Flag (F7425 from mouse and F3165 from rabbit, Sigma-Aldrich), anti-GAPDH (GA1R, MA5-15738, Invitrogen), anti-actin (ab8227, Abcam), anti-CycT1 (SC-10570, SCBT), anti-HEXIM1 (25388, Abcam), anti-CDK9 (SC-484, SCBT), anti-phospho CTD (Ser 2) (ab5095, Abcam). Membranes were washed five times and incubated with peroxidase-conjugated secondary Abs, which include ECL mouse IgG HRP-linked whole Ab (NA9310, GE Healthcare) and ECL rabbit IgG HRP-linked whole Ab (NA9340, GE Healthcare). After five washes, membranes were incubated in Western-Lightning Plus-ECL (Perkin Elmer) and visualized using the Odyssey Fc imaging system (Li-Cor).

Protocol tips
For detection of transiently expressed proteins, a total of 2.5 μg DNA, including pHT1-3 and/or pTat and/or empty pEF.Bos vector (see above), was transfected using Lipofectamine 3000 (Invitrogen) in 8.0E+05 293T cells. After 48 hrs, the cells were washed using PBS, and lysed using RIPA buffer containing 150 mM KCl and a protease inhibitor mixture (Thermo Fisher Scientific). Lysates were incubated for 10 min at 95°C in 2X Laemmli buffer (BioRad) supplemented with 5% DTT, and used for SDS-PAGE analysis in a 15% resolving gel. The proteins were then transferred to a PVDF membrane (BioRad), which was blocked in 5% milk in TBS and probed with specific primary Abs, which include anti-Myc (ab32 from mouse and ab9106 from rabbit, Abcam), anti-Flag (F7425 from mouse and F3165 from rabbit, Sigma-Aldrich), anti-GAPDH (GA1R, MA5-15738, Invitrogen), anti-actin (ab8227, Abcam), anti-CycT1 (SC-10570, SCBT), anti-HEXIM1 (25388, Abcam), anti-CDK9 (SC-484, SCBT), anti-phospho CTD (Ser 2) (ab5095, Abcam). Membranes were washed five times and incubated with peroxidase-conjugated secondary Abs, which include ECL mouse IgG HRP-linked whole Ab (NA9310, GE Healthcare) and ECL rabbit IgG HRP-linked whole Ab (NA9340, GE Healthcare). After five washes, membranes were incubated in Western-Lightning Plus-ECL (Perkin Elmer) and visualized using the Odyssey Fc imaging system (Li-Cor).

Protocol tips

For detection of transiently expressed proteins, a total of 2.5 μg DNA, including pHT1-3 and/or pTat and/or empty pEF.Bos vector (see above), was transfected using Lipofectamine 3000 (Invitrogen) in 8.0E+05 293T cells. After 48 hrs, the cells were washed using PBS, and lysed using RIPA buffer containing 150 mM KCl and a protease inhibitor mixture (Thermo Fisher Scientific). Lysates were incubated for 10 min at 95°C in 2X Laemmli buffer (BioRad) supplemented with 5% DTT, and used for SDS-PAGE analysis in a 15% resolving gel. The proteins were then transferred to a PVDF membrane (BioRad), which was blocked in 5% milk in TBS and probed with specific primary Abs, which include anti-Myc (ab32 from mouse and ab9106 from rabbit, Abcam), anti-Flag (F7425 from mouse and F3165 from rabbit, Sigma-Aldrich), anti-GAPDH (GA1R, MA5-15738, Invitrogen), anti-actin (ab8227, Abcam), anti-CycT1 (SC-10570, SCBT), anti-HEXIM1 (25388, Abcam), anti-CDK9 (SC-484, SCBT), anti-phospho CTD (Ser 2) (ab5095, Abcam). Membranes were washed five times and incubated with peroxidase-conjugated secondary Abs, which include ECL mouse IgG HRP-linked whole Ab (NA9310, GE Healthcare) and ECL rabbit IgG HRP-linked whole Ab (NA9340, GE Healthcare). After five washes, membranes were incubated in Western-Lightning Plus-ECL (Perkin Elmer) and visualized using the Odyssey Fc imaging system (Li-Cor).

Manufacturer protocol Publication protocol 1 Relevant paper

GAPDH Antibody

Proteintech Group

Upstream tips	Protocol tips	Downstream tips
	Western blot analysis was performed as previously reported17. The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA). GAPDH was used as control. The results were quantified by the ImagePro Plus 5.1 software (Media Cybernetics, Inc., USA).

Protocol tips
Western blot analysis was performed as previously reported17. The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA). GAPDH was used as control. The results were quantified by the ImagePro Plus 5.1 software (Media Cybernetics, Inc., USA).

Protocol tips

Western blot analysis was performed as previously reported17. The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA). GAPDH was used as control. The results were quantified by the ImagePro Plus 5.1 software (Media Cybernetics, Inc., USA).

Manufacturer protocol Publication protocol 1 Relevant paper

GAPDH Antibody (6C5): sc-32233

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Western blotting was carried out as described earlier (19). Briefly, the cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails (Thermo Fisher Scientific). The nuclear protein was isolated according to the protocol provided by the Nuclear Protein Extraction Kit (Thermo Fisher Scientific). Then, equal amounts of protein was resolved on 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). Membranes were incubated with primary antibody overnight at 4°C. Membranes were washed with TBST and incubated with horseradish peroxidase–conjugated secondary antibody. Proteins were visualized using ECL detection reagents (Beyotime Co.).

Protocol tips
Western blotting was carried out as described earlier (19). Briefly, the cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails (Thermo Fisher Scientific). The nuclear protein was isolated according to the protocol provided by the Nuclear Protein Extraction Kit (Thermo Fisher Scientific). Then, equal amounts of protein was resolved on 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). Membranes were incubated with primary antibody overnight at 4°C. Membranes were washed with TBST and incubated with horseradish peroxidase–conjugated secondary antibody. Proteins were visualized using ECL detection reagents (Beyotime Co.).

Protocol tips

Western blotting was carried out as described earlier (19). Briefly, the cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails (Thermo Fisher Scientific). The nuclear protein was isolated according to the protocol provided by the Nuclear Protein Extraction Kit (Thermo Fisher Scientific). Then, equal amounts of protein was resolved on 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). Membranes were incubated with primary antibody overnight at 4°C. Membranes were washed with TBST and incubated with horseradish peroxidase–conjugated secondary antibody. Proteins were visualized using ECL detection reagents (Beyotime Co.).

Manufacturer protocol Publication protocol 1 Relevant paper

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