4 replies to this topic

[s_liu]

Hi everyone,

I am running western blots to look for PKC. I recently changed my primary antibody to a new company-Sigma, and have been getting some "strange results".

Before, in a single lane, there are always several dark bands, with one band corresponding to PKC. The same applies to the rest of the lanes.

Now, in a single lane, there is only one band that corresponds to PKC and across all lanes, there is only this single band.

I have been using the same techniques before and after. So my question is, where have all the rest of the bands gone?

I also read the thread on how to reduce western blot background... Would you consider that with the new antibody, i have eliminated the background? as there is only my protein of interest left in one lane?

I am new to western blot technique, so please send any help advice and suggestions! I appreciate it very much!

Many thanks!!

[Simonsays]

The only thing I can think of is that maybe, I say MAYBE...
that your old antibody recognized PKC (autophosphorylated or not, which would give 2 bands) and that the new antibody recognize only the autophosphorylated form of PKC. This should be indicated on the antibody spec sheet.
Sorry I can't help you more.

Simon

[fred_33]

hi
as simosays tells in his message, check if your antibodies are recognizing the same epitope.

[bob1]

Hi

could it be that your original antibody was polyclonal and your new a monoclonal?

cheers
bob

[mabusheh]

s_liu, on Feb 14 2005, 10:47 AM, said:

Hi everyone,

I am running western blots to look for PKC. I recently changed my primary antibody to a new company-Sigma, and have been getting some "strange results".

Before, in a single lane, there are always several dark bands, with one band corresponding to PKC. The same applies to the rest of the lanes.

Now, in a single lane, there is only one band that corresponds to PKC and across all lanes, there is only this single band.

I have been using the same techniques before and after. So my question is, where have all the rest of the bands gone?

I also read the thread on how to reduce western blot background... Would you consider that with the new antibody, i have eliminated the background? as there is only my protein of interest left in one lane?

I am new to western blot technique, so please send any help advice and suggestions! I appreciate it very much!

Many thanks!!

if the new antibody is detecting your protein of interest, why worry about other bands? maybe the new antibody is not detecting other bands as u can see in ur results
the antibody reactivity differs from one to another. i.e. poly versus monoclonal.
if not happy with this antibody. go back to ur older one