Protein lysates were prepared by homogenizing samples of mesenteric PVAT in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Homogenates were centrifuged at 4°C, and the protein concentration of the lysates was quantified by Bradford assay. Protein lysate samples (10 µg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was immunoblotted with primary antibody followed by HRP-conjugated secondary antibody. Protein bands were detected by chemiluminescence using a ChemiDoc MP system (Bio-Rad Laboratories, Hercules, CA) and quantified using ImageJ software. Hypoxia-inducible factor-1α (HIF-1α) protein was probed using anti-HIF-1α antibody (ab179483, Abcam, San Francisco, CA). As a loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was probed using anti-GAPDH antibody (NB300-221, NOVUS Biological, Littleton, CO).
Anti gapdh antibody nb300 221
Manufactured by Novus Biologicals
The Anti-GAPDH antibody (NB300-221) is a laboratory reagent that binds to and detects the GAPDH protein. GAPDH is a commonly used loading control or reference protein in various experimental techniques. This antibody can be used to validate protein expression or quantify GAPDH levels.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Ab21667, by Abcam (1 mentions)
Ab120812, by Abcam (1 mentions)
Ccl22, by R&D Systems (1 mentions)
Recombinant human ccl2, by BioLegend (1 mentions)
Ab83250, by Abcam (1 mentions)
Anti akt antibody 9272, by Cell Signaling Technology (1 mentions)
Azd5363 s8019, by Selleck Chemicals (1 mentions)
2 protocols using anti gapdh antibody nb300 221
1
Mesenteric PVAT Protein Analysis
2
Evaluating Chemokine Receptor Signaling Pathways
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Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN). Recombinant human CCL2 was obtained from BioLegend (San Diego, CA). Antibodies used in western blotting, IHC, immunocytochemistry, and flow cytometry were as follows: rabbit anti-human CCR2 antibody (ab21667), rabbit anti-human CCR4 antibody (ab83250), goat anti-human CCR4 antibody (ab1669), rabbit anti-goat immunoglobulin (Ig)G H&L (fluorescein isothiocyanate; FITC) antibody (6737), goat anti-rabbit IgG H&L (FITC) antibody (6717), anti-mannose receptor antibody (ab64693), and anti-CCR7 antibody (ab103404) obtained from Abcam (Cambridge, MA); anti-Akt antibody (9272), anti-phospho-Akt (Thr308) antibody (9275), anti-phospho-Akt (Ser473) antibody (9271), and anti-rabbit IgG HRP-linked antibody (7074) obtained from Cell Signaling Technology (Danvers, MA); and anti-GAPDH antibody (NB300-221) obtained from Novus Biologicals (Littleton, CO). The antagonists used in the neutralizing assay were a selective CCR2 antagonist (ab120812) obtained from Abcam and a CCR4 antagonist (SC-221406) obtained from Santa Cruz Biotechnology (Dallas, TX). The Akt inhibitor AZD5363 (S8019) was obtained from Selleckchem (Houston, TX).
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