I made a western blot with the p65 NFkB antibody and the phospho p65 NFkB antibody from cell signalling. I get a band for both the phospho and non-phospho antibody at around 150kDa, how can this be explained?
hi
1-This band according to secondary antibody , you can re-probe your page and incubated again with primary antibody ( increase concentration ) and secondary antibody (decrease concentration) or run the new page
2- you can use from THP-I & MCF-7 as positive control
Note : this antigen" p65 NFkB " about 65-70 kd
https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol
good luck
Since p65 antibodies are well established and I guess that you use a reducing agent and an SDS-PAGE, I have to make the suggestion that you may made a misinterpretation with your size marker. I suggest to probe the membrane with a common antibody against a houskeeping protein of known size or use the next time an additional size marker.
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