@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..
I find suggesting PBS (phosphate buffer) for phosphoprotein detection an odd idea (even if it might occasionally work). 3% BSA (CST #9998) in TBS for blocking/antibody incubation is the best start, primary antibody is most influential on the outcome quality. Tween at 0.05 -0.1 % could be used to minimize the background.
Yes...BSA is preferable over milk for phosphorylated proteins in TBST
Lately I tried phosholipid binding proteins as analytes prepared in PBS (1% BSA) to be coated on ELISA plates and they gave huge background signals, suspicious regarding the phosphate and analyte interactions. Thus, in my opinion, TBS would be the best bet compared to PBS in this case.
as for blocking by milk, why only skim milk used in Western Blot ?
Jiashun: You don't want any fat sticking on your PVDF membrane, so people don't use whole milk or its powder.
A bit of historical "background" in Western Blotting involving anti-phosphate antibodies (please forgive me if you knew the story): in the old days, only anti-P-tyrosine or P-serine/threonine Abs were available, and blocking agents such as milk which contains a lot of P-proteins had to be avoided. Now, anti-P-protein Abs (most are monoclonal) recognize both the phosphate and its surrounding peptide as specific epitopes, and thus are not affected by free phosphate such as PBS or P-proteins with different sequences around. Therefore, use the cheaper milk powder (I buy from local Ralphs grocery store) is the best choice for clean background for us as consumers! :=)
Point taken, Ke-Wei, but phospho-proteins in milk can still be an issue. Some epitopes will be similar to your intended target, similar to background banding on a WB caused by spurious binding to other epitopes.
But milk has a couple of other limitations to consider.
Reproducibility: The box you buy from Ralph's today will be a different lot than the box you bought in October. How consistent is the manufacturing, and what characteristics are monitored? How much does protein, fat, and sugar content vary? Is everything homogenously mixed throughout the box? You may not want those sources of variability. Also, many people save and re-use diluted antibodies, particularly primary antibodies. In milk, those Ab solutions have a very short shelf life and will quickly spoil, even at 4C.
Loss of blotted proteins: A 1989 study demonstrated that soaking a blotted membrane in milk solution can cause loss of blotted proteins from the membrane. The effect is more severe with higher concentrations of milk. Just something to keep in mind.
A few points. TBS is probably preferable when using anti phosphor-aa antibodies, the phosphate in PBS probably wont hurt since as Ke-Wei says the epitopes also contain the amino acid, but why not get rid of phosphate. Its no harder to make TBS than PBS.
Historically, Ke-Wei is a bit off mark. The secondary antibody detection could be hooked to alkaline phosphatase, which with the right reagent gave a pink band on the blot. Obviously phosphate is an inhibitor of alkaline phosphatase and so that is why TBS was used. Now with chemiluminescence and IR imaging it doesn't make a difference.
Non-fat dry milk is used a) because it contains no fat, and b) it has an incredibly long shelf life.... lot longer than non-fat liquid milk. Try BSA to block, or fish gelatin or there are proprietary blockers. The big pain is that there is no universal blocker. You have to try everything with your primary and secondary antibodies to see which works best for your particular combination.
Neil's right - there is no universal blocker. You just have to try your antibodies and see what works best.
It is true that there is no universal blocker. I have used milk, BSA and commercially available blocking solutions. In my experience, blocking with milk gave equally good results as other blocking agents for both phospho and non-phospho proteins. But to increase the storage period of diluted antibodies, I used 1-2% BSA in TBST.
I block with 2.5% skim milk in PBS, apply primary and secondaries in PSBT (0.05% Tween), wash in TBST (0.05%) and have been using Phospho-AKT antibodies. I have had the antibody work fine, but the other day we ran out of PBS and so I instead used TBST (0.05%) to incubate my primary and secondaries. Suddenly the antibody no longer works AND some of the non-specific extra bands have sort of changed their profiles despite the same samples being run. Has anyone found that TBS inhibits their western blots? The TBS is a couple of years old (one of those 5L cubes of 20x), would this likely affect chemistry/concentrations etc?
If its not a transfer issue, its something else! Western blots do my head in!!
Ivana Marcia Alves Diniz
University of São Paulo
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