problems with housekeeping antibodies when re-probing membranes - (Apr/23/2012 )
I have troubles with the normalization of my WB. This is what happens.
1. I run, transfer onto PVDF (Millipore) and check with Ponceau S: equal loading
2. Probe with anti-tubulin and get signal corresponding to equal loading
3. Probe with Abs of interest and see accumulation of my proteins from 1st to last well, as I expected (I use from 1 to 4 Abs)
4. I probe with anti-tubulin or other Abs for housekeeping proteins and get the same identical signal as for my proteins of interest (I.e. I see accumulation of the housekeeping !!). This happens even if I don't perform step 2.
If I strip the membrane after my Abs of interest, the phenomenon is less strong.
What do you think can cause this phenomenon? And how can I solve it without stripping?
Thank you so much for any reply :-)
We need more detail of your blocking, incubation and washing steps to be able to help you.
antibody types: species of primary, species of secondary. concentration used and incubation time/protocol.
With the information you've given I'll just assume that you are still detecting the secondary antibody of your protein of interest when incubating with the house keeping antibody.
Also, what are the sizes of the proteins of interest. If the targets are not sufficiently different in size, I cant see how you can perform this without stripping.
Hi doc, thanks for your reply. Sorry for not being precise enpough. Here are the info you requested:
Blocking agent: 5% BSA in T-TBS (both used during the whole procedure as I'm working with antibodies against phosphorylated proteins). Blocking time: at least 30'/RT
Antibodies of interest: all rabbit antibodies from Cell Signaling (usually 1:1000 dilution as they suggest, but they don't say the initial concentration). They are unconjugated. Incubation time: ON/4°C
Housekeeping: all mouse antibodies
Secondary antibodies, again in 5% BSA in T-TBS, 1h/RT.
Detection method: ECL, usually imobilon from Millipore but sometimes ECL (normal/Plus) from GE.
Washes 3x10' between primary and secondary in T-TBS, plus an additional wash in TBS prior to developing.
Signal detection with VersaDoc.
Sizes: 15, 38, 50, 80 KDa for my proteins of interest. Housekeeping are 100, 55, 40, 36.
Hello again.I take it from your first post that you are not stripping your blots. Now, may I ask why? I don't see how you can distinguish between your protein of interest and your housekeeping ones if you are using ECL for all of them. If you don't strip the blots, the antibodies of your first incubation (say POI) will still be there when you are trying to detect the housekeeping ones. Seeing as some of them have very similar MW I really don't think you can differentiate them. Unless I'm missing something.
Also, if you are looking for phosphorylation, your "loading control / housekeeping" should be an antibody against the total protein independent of phosphorylation state (as far as I'm aware Cell signaling has matching antibodies for all the phospho ones). This way not only you control for WB loading, but you show that the phosphorilation increase (or decrease) you see is specific and not just due to higher or lower protein content. Again, for this I think you'd definitely need to strip your blots.
Maybe someone else here has a suggestion as to how to do this without stripping. The only way I can think of is using antibodies labelled with 2 different fluorophores, but cant really not think of a way to do it with ECL, sorry.