Urgent stuff about western blotting - (Nov/12/2006 )

Could anyone tell me what should I mix Primary antibody and secondary antibody with in the Western blotting? Should I use either TBS or TBS-T(Tween 20)?

TBS can be used for both primary and secondary Abs, although I use nonfat dry milk 3% for secondary Ab in order to reduce background.
Don´t use TBSTween, that´s the wash buffer.

Thank you for your advice. Should I add 1% milk powder in the Primary Ab solution?

I´ve never done that because background is often due to secondary Ab and so I´ve only added milk to the secondary. TBS will work well but probably 1% milk powder is not a bad idea. You should try and define the best conditions that work with your Abs.

I would advise you to check what the supplier suggests. There are several reagents to dilute your Ab with: I prefere to use 5%milk in PBS+0,1% Tween and 5%milkin TBS+0,1% Tween for anti-phospho-Abs. Anyway, some Abs work better in BSA.

Yes, I agree with dnafactory, in the datasheet should be described how to dilute your Ab.

If you have no indications, you have to try. You need to know that incubating your antibodies in TBS-T (with tween-20 0.05%) gives you less background, even for the primary.
If you don't have a good binding, then incubate in TBS.
you need to add a competitor : non fat milk, BSA, serum...
BSA blocks less than milk. the choice will depend on the affinity of your antibody, you have to try. I
Avoid milk if you detect phosphorylation or biotin. (because there are some in the milk)

Oh, I had been told not to dilute in TBST

we always dilute our antibodies (primary and secondary) with pbst (or tbst) with blocking agent.

oh what a surprise!