I am trying to probe for a loading control. I don't know why sometimes the bands show up and other times they don't. It's black or white whereas the actual protein I'm tagging for is consistent across replicates.
Any ideas? Or maybe it's just the antibody and I should try another control?
If the target protein antibody is so consistent why not just purify some target protein and use that as the control. Unless of course you want to control for loading levels in which case there are plently of tried and tested commercial antibodies available for this purpose (http://www.abcam.com/primary-antibodies/loading-control-guide).
Hi Zhou,
I LICOR, we have to play around secondary antibody concentration more than primary as I experience. While developing blots if you have higher background that masks our gene of interest also. Blocking reagent is crucial. I guess you are following the LICOR protocols for WB. In brief,
1)After protein transfered to NC membrane, wash with TBST and dry membrane for 1hr
2) Block 1hr with Blocking reagent provided by LICOR/Roche.
3) Incubate with primary 1hr/RT or ON/4degrees diluted in 3%Blocking reagent
4)Give 4-5 washes each 5min-10min with TBST
5) Add 1:20,000 secondary in TBST and incubate in RT for 1hr
6) Wash for 3-4 times with TBST and dry the membrane before going to developing
All the best.
Hi Chandra,
Thank you for letting me know. I will check those factors. I find however that it may be because the loading control I'm using (vinculin) is expressed at very low levels in my cell line of interest. That may be the reason why.
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