Hello all :)
I am choosing the primary antibody for WB analysis.
During look around, I found there is different predicted band size for this protein.
In same company, two primary antibodies are on the market.
However, the one showed the band around 40 kDa, while the other showed the band around 70 kDa.
Clonality (monoclonal), origin (rabbit) and tested cell line is same.
The only different thing is immunogen: full-length vs. 1-300 amino acid.
What is the reason for this different predicted band size?
and Which band is my targeted protein?
Hi So-Jin - if you have the primary amino acid seq then you can translate it and work out what MW the protein is predicted to be (as a quick check you can assume the average weight of each amino acid to be ~110 daltons thus if your primary seq is 170 amino acids the MW of the protein will be ~ 170 x 110 = ~18.7 kDa. (18,700 daltons).
Sometimes there is a discrepancy between the predicted weight and actual weight due to post-translational modifications such as glycosylation etc (so the protein sometimes runs bigger than it is predicted to be - it rarely runs a lot smaller than predicted so if you calculate that the weight should be ~70 then it is unlikely to be the 40 kDa one).
The second possibility is that the gene generates alternative splice variants..
Personally I would e-mail the ab manufacturers and ask why thye detect different sizes(as the ab are from the same manufacturer they should be able to answer this). Also check publications in the field and find out a) what proteins size is reported b) whether splice variants exist c) what manufacturer/ab is commonly used,
Sorry I can't be more help,
Gary
the antibody is the same. the investigated immunogen (probably transgenic cell extracts) are different. one contains the full -length protein, the other contains only the first 300 aas of your protein (a shorter construct), that is why appeares about 40 kDa. Check the pictures again.
Agree - antibody will be same. One will be to full length, other an alternate splice or shorter length
I agree with above: antibody sizes are often quoted in the context of a particular cell line with a particular post translational modification signature. Other lines may vary. In addition smaller bands can sometimes result from natural proteolytic cleavage of the protein or unnatural degradation in the lysate
some times fusion protein is able to find in cancer cell lines as EML4-ALK, ROS-ALK etc. The fusion proteins are also considerable. There is another, lysis buffer elements are important. Co-IP buffer is able to keep protein-protein interactions.
Western blotting is a technique that separates proteins based on size - in general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors and so the actual band size observed may differ from that predicted. Common factors include...
post-translational modification - e.g. phosphorylation, glycosylation etc which increases the size of the protein
post-translation cleavage - e.g. many proteins are synthesized as pro-proteins, and then cleaved to give the active form, e.g. pro-caspases
splice variants - alternative splicing may create different sized proteins from the same gene
relative charge - the composition of amino acids (charged vs non-charged)
multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands
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