Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

non-specific bands in western blotting - (Jun/06/2008 )

Hi,
I am getting a lot of nonspecific bands in my western blots, although my protein of interest is also visible. I do blocking with 5% milk powder made in TBST, overnight. Primary is used at 1:1000 dilution, made in TBST buffer. Can somebody tell me how to get rid of these non-specific bands. Is it a problem with the primary antibody or can it be overcome by changing other parameters
Mythri

-My3-

i would try to incubate the membrane only with the secondary antibody. sometimes you can be quite surprised about the result.....

-Ned Land-

QUOTE (My3 @ Jun 6 2008, 10:06 PM)
Hi,
I am getting a lot of nonspecific bands in my western blots, although my protein of interest is also visible. I do blocking with 5% milk powder made in TBST, overnight. Primary is used at 1:1000 dilution, made in TBST buffer. Can somebody tell me how to get rid of these non-specific bands. Is it a problem with the primary antibody or can it be overcome by changing other parameters
Mythri


may be 1:1000 dilution is not enough; try higher dilutions (1:2000, 1:5000); or sec. Ab is to high (standard dilution is 1:1000);

re-use the first Ab, after 2- or 3-time usage, background may decrease

-The Bearer-

QUOTE (My3 @ Jun 6 2008, 11:06 PM)
Hi,
I am getting a lot of nonspecific bands in my western blots, although my protein of interest is also visible. I do blocking with 5% milk powder made in TBST, overnight. Primary is used at 1:1000 dilution, made in TBST buffer. Can somebody tell me how to get rid of these non-specific bands. Is it a problem with the primary antibody or can it be overcome by changing other parameters
Mythri

1) First check if the non-specific is due to primary or secondary by incubating your immunoblot with the secondary antibody. With the secondary antibody alone, if you are still getting the same non-specifics, then you know where you need to fix it - either reduce the contact period with the secondary or increase the washings
2) If you determine that the non-specific is due to primary, try increasing the blocking period (5% milk will be fine) to overnight and incubate with primary for shorter period (say, 1hr).
Let me know if it worked.
Good luck.
Satya

-newresearcher-