I was wondering if anyone has tried making blocking solution (5% donkey serum) in PBST and also in PBS and looked at immunostaining results. From my western blot knowledge we don't make blocking solution in any detergent so any suggestion will be great.
Detergents tend to inhibit protein interactions, in the case of blocking it would tend to inhibit the interaction of your serum or milk protein with the membrane. Since this is generally the opposite of what you want with blocking (you want your blocking protein to bind up all the non-specific interactions on the membrane), it's not common to add detergent in the blocking solution, though I suspect a bit of Tween doesn't make a lot of difference in most cases.
I had the same impression that we don't use detergents in blocking. not sure why my lab protocols include using TBST in blocking solution
Hi Abbas,
I always use TBST for all steps of my westerns and I never had any problems. On the contrary, I have seen high backgrounds if not used (I used 0.1% Tween 20, 5%Blocking reagent in TBS). I think if you go higher in the detergent concentration only then you may not be able to block the nonspecific sites. I can give you my protocol if you wish.
@Aydan: I am still confused why we need a detergent in blocking solution. If anyone has done it with and without detergent and getting the same results then why use it.
the rest of washing/primary/secondary can be done with TBST but not the blocking step. what do you think?
Hi Abbas,
like Aydan I have always used Tween-20(0.1%) for Western Blotting and Triton X-100 (0.05%) for Immunohistochemistry in all my blocking and antibody diluents. I only do the very last wash (prior to ECL detection) in TBS alone.
I have never blocked without detergent when doing WB, and I suppose I use TBS-T (with skim milk powder/serums) out of convenience, because after blocking the membrane I will then just add the 1oAb to the blocking solution - economising :)
good luck
Well, first TBST is TBS buffer with Tween20. A bit of detergent in my hands help to get a clear background. Especially if you are doing chemiluminescence, dirty background will give you big trouble. In immunostaining you surely want to block the nonspecific sites but also clean up the excess substrates and blocking reagents. If not, the secondary Ab can bind nonspecifically to all over giving you a dirty background.
I would suggest go with 0.1% Tween20 (TBST). I never did myself without Tween20 but seen westerns done by others and didn't look so clean. As you wish you can give it a try. If you make long washes in between steps may be you can get a good look. Try it!
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