Hi

if you are investigating membrane associated proteins, you should also try to not boil your protein samples in laemli buffer, but instead you could let them at RT for a while (around 10 minutes for example) after laemli buffer addition, and load your western blot gel. I had a similar problem when I was blotting endogenous CXCR4 in HEK293T cells. If your protein of interest has different transmembrane domains (in particular beta-sheet structures), the boiling at 95-100 °C in lameli buffer could cause severe protein aggregation which will not allow your proteins to migrate in the gel (do you see any unexpected band at the top of your gel? if yes, the non-boiling will solve the problem).

Probably, the band you see could just be an unspecific band caused by your antibody (especially if you use the mouse antibody anti-FLAG from SIGMA, which gives a lot of unspecific bands)

Hi Andrea,

Thanks for the comments. I never boil my samples since they are membrane proteins. I usually let them keep at 37°C for 30 min. My proteins are visible at ~130KDa and yes I can see some unexpected bands at top. Specially, when I use three or more passages of HEK, the possibility of getting band rises. Have you solved your issue by fractionation?

Thanks

If you see bands at the top in your overexpressing samples is because protein are not migrating. I would try at this point either to boil the samples or fractionation. I did not try fractionation in this case

Hello Andrea,

Let me clarify more on that. My proteins are adenylate cyclase (AC) transfected in HEK cell. I am using Flag antibody from Abcam. My ACs are tagged with Flag. At first I used dry-milk as blocking solution but it masked my proteins and then BSA, but it did not help me as well. Afterwards, I went through fish-gelatin blocking and it perfectly worked. But I have another problem now which is band for HEK (negative control) exactly at the same lane of my proteins. I suspected of cross-contamination but I used another HEK cell from another lab which also had band. My proteins have band at around ~130 kDa. I attached the blot.

Andrea Emanuelli

Hi

without protein ladder is hard to interpret your blot. I think that band is just aspecific coming from FLAG antibody. Did you try to probe the membrane with anti-AC antibody? By the way, is it normal that you have different patterns of migration in your infected cells? And have you sequence verified your plasmid(s)? just to be sure to not have unpleasant surprises from your DNA sequences of the plasmids...