Hello Temitope

The RIPA buffer content seems to be fine. The cells have to be incubated in RIPA buffer on ice for 5 minutes. All the following steps have to be carried out at 4 degrees centigrade. The scrapping of cells has to be done on ice, The centrifugation of the lysate has to be done at 4 degrees centigrade for 10 mins at 8000g. The supernatant containing the soluble protein must be transferred to the tube kept on ice. Also recheck your culture conditions for hypoxia induced NFkappaB in MDA MB 231.

Also NF kappa B p65 antibody is good for the detection of specific phosphorylated protein expression in many cancer cell lines.

Thank you.

Hello!

Maybe you will be interested in this article:

Article Collateral Sensitivity of Parthenolide via NF-κB and HIF-α I...

SDS-PAGE and Western Blot Analysis

Thirty mg/ml were taken from the protein fraction, and SDS-loading dye was added following by heating at 95°C for 10 min. After the denaturation process, proteins were loaded onto 10% SDS-polyacrylamide gels. A Western blotting apparatus was used to transfer proteins on a PVDF membrane (Roti® PVDF, pore size 0.45 μm, Carl Roth GmbH, Karlsruhe, Germany). The membrane was blocked using 5% BSA/TBS-T and then incubated with primary antibodies against NF-κB p65 (D14E12), IκB (44D4), HIFα (D2U3T), or β-actin (13E5) overnight at a dilution of 1:1000. HRP-linked secondary anti-rabbit antibody (1:2000) was then added and incubated for 1 h. Both primary and secondary antibodies were purchased from Cell Signaling (Frankfurt am Main, Germany). Luminata Classico HRP Western Blot substrate (Merck Millipore, Schwalbach, Germany) was used for the detection step and membranes was visualized with aid of Alpha Innotech FluorChem Q system (Biozym, Oldendorf, Germany)

maybe you can try to use a phosphatase inhibitor cocktail. I use RIPA buffer from Cell Signaling. It works for me for the detection of Phospho-ERK.