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a question about western blot - question and help western blot (Jun/20/2005 )

Hi all,

I am new to western blotting and i have a question about the procedure. is it possible to use two different primary Ab in the first step (both Ab suppose to react with different size protein -30kDa and 75 kDa) their 2ndry Ab is the same.
can i save reprobing doing this?

right now the procdure i am following is
1. 30 min block 4% milk
2. 1.5 hrs 1 Ab 1:10000
3. 4*10min wash pbs-t
4. 50min 2 Ab 1:20000
5. 4*10 min wash
6. 5 min ECL
7. exposure to film

thanks for your comments


nope. sometime you may have non-specific binding and it may be difficult to differentiate between the bands. Thatz why u have ur MW marker. I think the best way is to add one antibody at a time and then reprobe.

my protocol is as follows:
5% milk in TTBS one hour RT
primary in milk (dilution depends on the company) usually overnight 4C
wash 3X 5 mins TTBS
secondary in milk 1 h RT
wash 3X 5 mins TTBS

good luck


i don't know whether you can use two primary ab at the same time, but i am sure you can save the membrane for reprobing. there is a very good protocol from Amersham Bioscienses, which tells you a lot about western blot and reprobing. i cannot remember the website now. just go search. it tells you everything and very very useful!


I probe for p21 and p27 at the same time as they both use the same secondary. I never see much background however you may want to try it separate on one blot and then together on another blot and see if it makes a difference as far as background and non-specific binding goes.


hi! i agree with pria that you may have unspecific binding if you incubate primary and secondary togehter. but i've never really tried it. i normally block the membrane overnight with fat free milk in tbs tween, and then incubate the next day wth primary antibody for 1-2 hours, wash 4x with tbs-tween, 1-2 hours of sec antibody incubation, wash again 4 x usually 10 min long washes. but time of incubation depends on the sensitivity of the primary antibody and how much protein you load.


Considering that the two proteins are separated that far apart, there is no reason why you con't put both primarys on at the same time. So long as background for both of them are low when used separatly, it should be low when you put them in together.

I have successfully done this when the two proteins were 50kD and 35kD. It is a great way to save time.