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Western blot mystery


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#1 eleceyes

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Posted 11 January 2005 - 02:17 PM

Just a quick question about interpreting bands appear on a western blot:
On a western blot, I often see multiple bands, i.e. band running at the expected size (theoretical size) as well as bands at other places.
I was told/taught to ignore the bands which appear at places other than its 'theoretical size' because they are 'unspecific'.
It worries me a bit because the intensity of these 'unspecific' bands sometime is just as intense as the 'specific' band. They could well be the products of post-translational modification (or even different degree of post-translational modification or protein degradation) or formation of dimers etc etc.

I was wondering how should I go about explaining these bands?

Thank you millions in advance for answering the question.

#2 scientist

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Posted 13 January 2005 - 12:41 PM

We have the same issues in our lab as well and I have come to the following conclusions....

1. the antibody is not specific and cross reacts with other proteins

2. the protein has different isoforms (do you know anything about this?)

3. The protein binds to other proteins making it larger

The definitive would be mass-spec on one of the larger bands to determine if your peptides are present with other unknown peptide.

Can you induce expression or inhibit the expression of your protein? If so, do you see an increase or decrease in the other bands as well?
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#3 manojdarolia

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Posted 20 January 2005 - 05:52 AM

hi

You didnt mentions the size of the extra bands and how many bands are there.

well if the antibody is crossreacting, that can be checked by putting some control with it if some control is possible.

also if the protein is binding to some other protein, those interactions can be broken down by using some reduing agent ,

also may be the case of protein aggregation, also the reducing agent can be used for that




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