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Western Blotting. - How to reduce non-specfiic bound signals?? (Aug/12/2002 )

Dear all,

I'm a new bird in doing western blotting. I'm now doing a western blotting for the membrane protein on culture cell. After immunostaining with my primary antibody and secondary antibody, the positive signals were visualized by using ECL+ and expose for 3 minutes. Eventually, I found that there are lots quite a number of no of non-specfic bands and their abundance increase with time. I don't what the problems. I here quote the procedure for my washing and hope there is somme comment on that:

Step 1.Wash twice (5 minutes each) with Washing Buffer A
Step 2.Block the blot for 2 hours at room temperature with Blocking Buffer
Step 3.The blots to be incubated overnight at room temperature with the same blocking solution containing 3mg/mL of 1˘X antibody.
Step 4.The blot to be then washed 5 times (5 minutes each) with Blocking Buffer.
Step 5.Then, incubate the 2˘X antibody of 1:5000-fold dilution in Blocking Buffer for 1 hour at room temperature.
Step 6.Then, the blots to be washed in twice (5 minutes each) with Washing Buffer A.
Step 7.Then, the blots to be washed with Washing Buffer B twice; 5 minutes and then followed by 10 minutes.
Step 8.The blots to be washed with Washing Buffer A for 5 minutes
Step 9.Then, the blots to be washed with Washing Buffer B for 10 minutes
Step 10.Finally, the blots to be washed with Washing Buffer A only.

Million thanks!


Hi Daniel,
how sure are you with your antibody? If you see clear bands and not a smear they seem to be for real. Maybe you can try to purify the antibody. But you will need a specific antigene for that.



to prevent ur non specific binding u need to block ur celllose membrane atleast for about 45 mts - 2 hr using a good blocker...i use 5 % non fat dry milk to blekc the memebrane before i use it for doing my testing.if u havent then u should block it.after blocking u can store it and then use it or maybe u can use the membrane after works really well and should get off ur wrong signals.
the secind possibility is that ur wash buffer.i dunno what wash buffer u use.i use 1x TBS and 0.3% tween but u can vary the tween amoiunt depending upon ur antigen concentration...(0.05%-0.3%).washing is done twice or thrice after secondary antibody treatment.u need to wash it then with 1% TBS before u add ur substrate.


maybe your secondary antibody produces the unspecific signals. try another one.
another possibility is to use not the whole cell extract but only the membrane fraction.



I think your primary Ab concentration might be too high. Is the primary Ab 3mg/ml the stock concentration? Because if 3mg/ml is the diluted concentration then that is insanely high. The diluted concentration should be in the ug range. If the stock is 3mg/ml I would recommend a 1:1000 dilution at least.



u are using a very high concentration of primary antibody and also ur incubation with primary antibody is extremely high. in my opinion for a non-perified antibody of inferior binding capacity 2hrs incubation is more than sufficient. use abot 1:2000 of the antibody and incubate for not more than 2 hrs. may be this should help.
all the best


IĦĦthink the concertration of secondary antibody is too high. The unspecific bands must be produced by the secondary antibody. You can dilute more of the secondary antibody. Another possibility is that u may decrease the expose time.

Good luck!