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Gapdh mab

Manufactured by Cell Signaling Technology
Sourced in United States

GAPDH mAb is a monoclonal antibody that targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in glycolysis, playing a crucial role in cellular energy metabolism. This antibody is commonly used as a loading control or reference protein in Western blotting and other immunoassays.

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6 protocols using gapdh mab

1

Western Blot Analysis of Bim Protein

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Proteins were extracted from cells using ice-cold RIPA lysis buffer containing 1 μM phenylmethanesulfonyl fluoride (PMSF; Sigma). Equal amounts of protein were separated by SDS-PAGE and blotted onto PVDF membranes. After blocking, the membranes were probed with an anti-Bim mAb (#2933S; 1:1000 dilution; Cell Signaling Technology, Beverly, USA) overnight at 4°C. A GAPDH mAb (#5174S; 1:1000 dilution; Cell Signaling Technology) was used as the control. Then the PVDF membranes incubated with HRP-conjugated secondary antibody (1:10000 dilution; Cell Signaling Technology) for 1 h at room temperature. The protein bands were visualized using the Enhanced chemiluminescent (ECL) detection reagents (Monad).
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2

Protein Expression Analysis Protocol

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Cells or tissues were lysed in RIPA buffer. The following primary antibodies were used to detect the proteins of interest: (1) cyclin antibodies (Cyclin Antibody Sampler Kit, Cell Signaling, Danvers, MA, USA); (2) mTOR mAb, 4E-BP1 mAb, p70 S6 kinase mAb, and GAPDH mAb (Cell Signaling, Danvers, MA,USA); (3) mouse GFAP mAb and rabbit DCX polyclonal antibody (Santa Cruz, Dallas, TX, USA). Protein bands were visualized and optical density was analyzed using a Biospectrum 500 imaging system (UVP, LLC, Upland, CA, USA).
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3

Western Blot Analysis of Antigen Presentation Proteins

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Proteins were extracted from 1 × 107 cells from the three HNSCC cell lines and protein concentration was determined with the Pierce BCA protein assay kit (Fisher Scientific, Schwerte, Germany). For Western blot analyses, 50 μg protein/lane was separated on 8%-12% SDS-PAGEs, transferred onto nitrocellulose membranes (Schleicher and Schüll, Dassel, Germany) and stained with (3%, w/v) Ponceau S. Immunodetection was performed with specific primary mAbs directed against TAP1, TAP2, LMP2, LMP10, HLA-I HC and β2-m (kindly provided by S. Ferrone, Harvard, Boston, MA, USA) as recently described [8 (link)]. Staining of blots with a GAPDH mAb (Cell Signaling, Frankfurt, Germany) served as loading control. As secondary antibodies the horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies (DAKO, Hamburg, Germany), respectively, were used, and the LumiLight WB substrate (Roche Diagnostics, Mannheim, Germany) was employed for detection and visualization with a CCD camera (Eastman Kodak Co., Berlin, Germany).
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4

Profiling Xenobiotic Stress Responses

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Cell pellets corresponded to untreated cells in media, 0.1% DMSO, B[a]P treated (1 μM), DATS treated (40 μM), and CoTx (1 μM B[a]P combined with 40 μM DATS) after 24 h treatment. 0.5% TritonX-100 was mixed with a protease inhibitor cocktail and combined with each pellet. The Pierce BCA Protein Assay kit was used to determine protein concentration. Each sample contained 50 μg of protein. 1:1000 dilution of primary antibody and secondary antibody were used. After the secondary antibody was incubated, the protein was identified, and the digital immunoblot was taken. The primary antibodies tested were CYP1A1 (ab235185) purchased from Abcam (Boston, MA, USA), Hypoxia Pathway Antibody Sampler Kit (#15792), AhR mAb (#83200), and loading control GAPDH mAb (#D16H11) purchased from Cell Signaling (Danvers, MA, USA).
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5

Western Blot Analysis of Protein Samples

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Protein samples were subjected to gel electrophoresis, transferred to a PVDF membrane and blocked with a 5% wt/vol BSA PBST (0.1% Tween-20) solution for 1 h. Membranes were incubated with primary antibodies overnight at 4 °C; MMP3 pAb diluted 1:1,000 (Proteintech Group, 17873-1-AP), GAPDH mAb diluted 1:1000 (Cell Signaling Technology, 2118S). Membranes were washed with PBST (0.1% Tween-20) and incubated with secondary antibodies for 1 h at room temperature; horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody diluted 1:2,000 (Thermo Fisher Scientific, G-21234). Membranes were washed with PBST (0.1% Tween-20) and imaged with a chemiluminescence reagent (Thermo Fisher Scientific, 34095). Densitometry analyses were performed using ImageJ software.
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6

Western Blot Quantification of MMP3 and GAPDH

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Protein samples were subjected to gel electrophoresis, transferred to a PVDF membrane and blocked with a 5% wt/vol BSA PBST (0.1% Tween-20) solution for 1 h. Membranes were incubated with primary antibodies overnight at 4 °C; MMP3 pAb diluted 1:1,000 (Proteintech Group, 17873-1-AP), GAPDH mAb diluted 1:1000 ,j
(Cell Signaling Technology, 2118S). Membranes were washed with PBST (0.1% Tween-20) and incubated with secondary antibodies for 1 h at room temperature; horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody diluted 1:2,000 (Thermo Fisher Scientific, G-21234). Membranes were washed with PBST (0.1% Tween-20) and imaged with a chemiluminescence reagent (Thermo Fisher Scientific, 34095). Densitometry analyses were performed using ImageJ software.
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