HIF-1α Antibody (28b): sc-13515

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Treated ECA109 cells were lysed in SDS Lysis Buffer (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and then centrifuged at 14,000 × g (15 min, 4°C). Protein concentrations of the samples were calculated with a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Equal amounts of protein were separated by 6 or 10% SDS-PAGE, transferred to Protran® nitrocellulose membranes (Schleicher & Schuell BioScience GmbH, Inc., Dassel, Germany), and then blocked with TBS (pH 7.4) containing 0.05% Tween 20 and 5% nonfat milk. The membranes were incubated overnight at 4°C by gentle agitation with various primary antibodies: Anti-AKT antibody (1:500), anti-p-AKT antibody (1:500), anti-HIF-1α antibody (1:500), anti-VEGF antibody (1:250) and anti-β-actin antibody (1:250). The following day, the membrane was incubated with alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (Ig) G or goat anti-rabbit IgG as secondary antibody (1:2,000; BS13271; Bioworld Technology, Inc., St. Louis Park, MN, USA) for 1 h at room temperature. The blots were visualized using the SuperSignal West Femto kit (Pierce; Thermo Fisher Scientific, Inc.).

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Manufacturer protocol

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