extra bands on western blots

Frank O. Fackelmayer Frank at Fackelmayer.de
Wed Apr 10 07:15:28 EST 2002


Hi Sophie,
there is a multitude of possible reasons for your problem, so it is hard
to give specific advice.

1. how specifically does your antibody recognize your protein in its
natural surrounding (total cell lysate from the source cells)?

1a. did you titrate the amount of antibody to get a specific signal?
When the antibody is a commercial one, try using MUCH less than recommended...

2. does the antibody show bands in the lysate of bacteria that do not
have your plasmid and hence do not make your protein (suggesting there
are crossreactive bacterial proteins). 

3. Do the additional bands increase in intensity when you induce protein expression?

4. What size do the additional bands in the bacterial lysate have? Are
they smaller than the protein you want (suggesting degradation in the
bacteria or during purification)? 

5. when they are bigger, and you do not see them in nonexpressing
bacteria: did you sequence your construct to make sure it has the
correct reading frame and a stop codon at the end? 

6. Is your protein known to be "sticky", eg. because it is a membrane
protein that aggregates even (or especially) upon boiling in SDS sample buffer?

7.....



best regards,
Frank




Sophie wrote:
> 
> I am looking at the expression of a cloned protein in a number of different
> bacteria and I keep seeing quite a lot of extra bands on my western blots.
> Can anyone suggest why they occur and how I can get rid of them?
> 
> Thanks,
> 
> Sophie.
> 
> <http://www.biowww.net/forum/read.php?f=6&i=992&t=992>




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