Hi Muhammad,

I don´t really have experience with ELISA, but for WB I always use just TBS/Tween-20 for blocking and this works nicely!

Hope it helps,

good luck!

Thanks Anita 

you use only BS/Tween-20 for blocking? means with BSA or Skimmed Milk or fish gelatin?

I need such a recipe which should be protein free blocking buffer (an alternate of commercially available Thermo Scientific Pierce Protein-Free (PBS) Blocking Buffer)  as I am working with mammal proteins and their is a chance of cross reactivity if we use animal protein based blocking buffer (i.e. BSA-PBS or Gelatin-PBS or any other like these buffer)

TBS is Tris-buffered saline. Tween-20 is the blocking agent.

Tween-20 is a detergent. Its used for washing the membrane. I don't think its effective in blocking. 

Unfortunately I don't know an alternative.. in a quick google search I found something using Polyvinyl alcohol. 

Check this:

http://www.ncbi.nlm.nih.gov/pubmed/19247640

Muhammad,

I use only TBS/Tween-20, without any proteins added.

Yes, Tween-20 is a detergent and it can work for blocking. You would probably have to test it for your antibody-antigen combination, but I think it´s a cheap alternative worth a try.

Thank you very much Anita, I am trying to use TBS/Tween-20, 

does TBS/1%tween-20 work better? or some different ratio of tween-20 is recommended

I use 0,1% Tween- same as for washing after antibody incubations. It works fine for me.

Thank you Anita. Going to follow it :)

A protein free Blocking buffer can be made from 2% sucrose, PBS.  

You could use Tris-buffered saline (TBS) or PBS  with 1% or 0.5% Ficoll 400 for blocking.