HIF-1 alpha Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (1:200) -Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Protocol tips
-Rabbit (1:200) -Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Publication protocol

Protein extractions were performed from tumour homogenates in lysis buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% sodium dodecyl sulfate, 1% NP-40, 0.5% Na-deoxycholate and 5 mm EDTA supplemented with the protein inhibitor cocktail Complete Mini, EDTA-free (Roche Diagnostics, Indianapolis, IN, USA). Cellular debris were cleared by centrifugation and supernatants were aliquoted and stored at −80 °C for further use. Protein quantification assay was performed with a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). The protein extracts were applied on a 12% polyacrylamide-SDS gel at 120 V for 3 h at 4 °C and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA) using the Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA) settled at 160 V for 1 h. The membrane was blocked with 8% reconstituted skim milk powder in TBST solution (10 mm Tris–HCl pH 7.5 containing 150 mm NaCl and 0.05% Tween 20). The blots were incubated with MT1-MMP (1 : 750, sc30074, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or HIF-1α antibodies (1 : 200, NB-100-654; Novus Biologicals, Oakville, ON, Canada) in blocking solution overnight at 4 °C. After washing with TBST, rabbit horseradish peroxidase-conjugated secondary antibodies (1 : 10 000, LS-C181152, LifeSpan BioSciences, Seattle, WA, USA) were applied and the blots developed by the Enhanced Chemiluminescence Detection System (Perkin Elmer, Waltham, MA, USA). Relative intensity of the bands were normalised to α-tubulin internal standard.

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Manufacturer protocol

Download the product protocol from Novus Biologicals for HIF-1 alpha Antibody below.

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