Samples were anti-CD3 and anti-CD28 co-stimulated Jurkat cells. The gel was 10% acrylamide SDS-PAGE. Semi-wet transfer to nitrocellulose membrane using TransBlot Turbo system. Membrane was incubated with 1:1000 Phospho-NF-kB p65 (Ser536) (93H1) Rabbit mAb and · GAPDH (D16H11) XP Rabbit mAb overnight. GAPDH was used as the loading control. Secondary antibody was Goat anti-rabbit IgG HRP.
Membrane visualised using SuperSignal West Pico PLUS. Phospho-NF-kB bands were visible on the membrane along with the ladders but GAPDH bands were only visible for two of the 9 samples loaded - the rest were blank for GAPDH. I can't figure out why this happened - I initially suspected too low concentrations of GAPDH but then I thought this would not be the case as there was binding of the GAPDH to at least two of the samples.
Any suggestions or guidance as to where to look to figure this out would be greatly appreciated.
Did you measure the protein concentration and loaded equal amounts of total proteins? You can try to stain the membrane and make sure that the proteins are loaded appropriately in all the lanes and show constant levels. This is now accepted as a loading control. One possibility is that the GAPDH is regulated in your cells which happened to me before and you need to shift to another one.
I attached this link for your reference:
Article An appropriate loading control for western blot analysis in ...
Thanks for your suggestion Sherif. Yes - the protein concentrations were measured via nanodrop - and same concentrations were loaded in each well of the gel. That is a good point about the possibility of regulation of GAPDH - thank you! I appreciate your help with this.
Hi,
you should stain your membranes with Ponceau S stain to confirm an equal loading amount. Another possibility is that GAPDH was upregulated in those 2 samples making the signal in other lanes faint but I have never encountered this effect with housekeeping proteins (GAPDH, Actin, Tubilin, Vinkulin). You can try some other housekeeping protein to confirm.
Thanks Mateo. So I re-incubated with rat cyclophillin-A as the primary and anti-rat as secondary for 1hr at room temp. This showed very strong bands on the same membrane. This still makes me confused about GAPDH and why that didn't show up.
- Badges
- Science topic