@Farhan: In my experience it doesn't matter if you do this at room temperature or at 4°C. Shakers are usually located in the cold rooms, so I did this there.
I don't work with phosphorylated proteins but I saw milk in TBS-t leave the Western blots, high background, all black. So Dr. use BSA in TBS-T and low background, now at least the protein signals were seen, before anything. @Farhan the tempeture, as like Christian sais, it's dosen't matter, I blok with milk one hour a room tempeture.
I use PBST along with BSA 5% for phosphorilated proteins, it works very well.
I have been working with phosphoproteins as well. I found that even blocking with milk at room temperature for an hour works perfectly fine as long as you do at least 3 washings with TBS-T before putting your primary dissolved in BSA. Then every antibody can be different, but even the manufacturer was suggesting to use milk for blocking
Francesca Pieropan,What concentration of Tween-20 are you using in your TBS-T buffer?
Hi Susana, I am using 1% Tween-20 in TBS
I have used milk for blocking. If you get high background, I suugest to increase the number of washing steps with PBS-T.
I have used 2% BSA in TBS-T for blocking and 0.2% BSA in TBS for 1o antibody as well as 2o antibody and got nothing in one blot and another blot got dark completely. What is the most used % of BSA in both purposes?
I have usually used 5%milk in 1%Tween -20 TBS as blocking reagent and 5%BSA 1%Tween-20 TBS as diluent for phosphoantibody.
However, I have recently bought a phosphoantibody of which the package insert suggested to use 5%milk as blocking reagent as well as diluent for antiboby.
I had contacted the technical support and was confirmed that this particular phosphoantibody has worked well in milk. In addition it is a mouse monoclonal so it is almost always recommend for use in 5%milk. I know about the phosphocasein in milk but don't know about the mouse monoclonal.
I will try both BSA and milk since I usually do WB in duplicate, and see which one would give a better result.
So there might be a possibility that milk would work with phosphoantibody.
I have tried both BSA as well as milk. In my hands milk works best for me for all the antibodies. I dont think TBST or PBST make huge difference. You can also try fish serum as blocking agent. Its expensive but you will get very clean blots with all the antibodies.
Most of the phosphoprotein antibody kit suggest to use BSA as blocking agent but some time some phosphoproetin also work using milk so you try by using both. if does not work may be some other problem. May be the amount of protien you load is less. generally in the case of phosphopreotin we use higher amount of protein.
Its always good to use Tween to reduce the background. But don't use it when you are adding the Abs since it reduces the signal.
safest fist bet is WB-dedicated BSA from CST(albeit expensive), Tween is used at 0.1% (not 1!), and might sometimes be omitted from Ab solution. Some Abs work better at RT (not anti-P forms though)
For Phospho- proteins BSA works well with PBS or PBST depending upon your antibody sensitivity.
We routinely use 5% skimmed milk in TBS-T for Westerns. We only use 1% BSA instead when there is a problem (high background etc) in using milk for detecting protein phosphorylation..
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