GAPDH Loading Control Monoclonal Antibody (GA1R)

Protocol tips

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	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).

Publication protocol

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).

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