i am using a polyclonal rabbit primary for detecting a 50KDa protein but keep getting nonspecific band around 30KDa and few others.
- I block using 5% non fat milk for one hour
- sample preparation by heating at 80C for 10-15 minutes.
Hi Revathy,
I got the same problem a few years ago. I cut the nitrocellulose membrane above my protein size (or below, depending on the size of nonspecific protein) and only incubated that piece of membrane with my antibody. Doing this you avoid the binding of you antibody to the non-specific protein.If you don't want to cut your membrane you can try increasing concentration of milk up to 8% in TBS buffer.
Good luck!
You may first try immuno-precipitation during sample preparation and then do western blotting. It might help you in determining your required protein.
Usually, polyclonal Abs give this problem. It will be a lot easier to use a monclonal Ab.
You may also first screen your sample on SDS and cut your protein of interest from the gel, solubilize and proceed with the WB.
Hi Revathy,
I got the same problem a few years ago. I cut the nitrocellulose membrane above my protein size (or below, depending on the size of nonspecific protein) and only incubated that piece of membrane with my antibody. Doing this you avoid the binding of you antibody to the non-specific protein.If you don't want to cut your membrane you can try increasing concentration of milk up to 8% in TBS buffer.
Good luck!
Hi there!
Increasing the amount of Tween-20 and blocking agent, as Carmen suggested, may help.
Bests!!
Hi there,
Increase the time of blocking, for example 2h with 5%milk @RT or overnight @4 degrees.
Good luck!
It is likely that the 30KDa band is not so much non-specific as cross reactive, or even a breakdown product of the 52KDa protein. Or, your polyclonal antibody may have been raised against an impure antigen. The Igs reacting with minor bands may be truly non-specific (i.e. "naturally occurring"), however, and in that case should be present at a lower titer than antibodies against your protein of interest. Dilute your primary antibody and see if that helps. This approach depends on the purpose of your immunoblot (e.g. are you following a purification or trying to show an increase in specific protein expression following some treatment?). Think carefully about this.
It might be helpful to dilute your secondary antibody further, as well. What detection method are you using? i.e. enzymatic/colorimetric, chemiluminescent, far IR? If one of the first two options, be sure you are not letting the reaction proceed too long. Also, if at all possible, a control blot with either pre-immune or no primary antibody would be helpful in diagnosing the genesis of the problem.
5% milk should be plenty for blocking, but do include 0.05-0.1% Tween 20 in all washes and antibody dilutions (in the same % milk).
Sometimes addition of 10 % horse serum during incubation with the primary antibody helps.
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