my protein is 18Kda but runs as a 25kda on WB - why ? - (Aug/02/2006 )
Hi,
I'm working on a protein which supposes to be ~18Kda (by calculation 169aa). How ever in te WB it look like a ~ 25Kda band (the band is quite sharp NOT diffused).
I'm thinking the reason is post translation modification.. but which one can add 7Kda ???? (Someone told me that a sharp band exclude glycosilation or Ubiqintion).
any advise will be most welcomed (as to a possible reason and a method to examine that...)
THANKS !!!
Lior.
just to confirm - you aren't just using a prestained marker to confirm your size?
some of these are notoriously bad for sizing - we used to run one of these as a transfer check, and also an unstained (way more accurate) one which we then post stained with coomassie......
sorry if you already know this!
some of these are notoriously bad for sizing - we used to run one of these as a transfer check, and also an unstained (way more accurate) one which we then post stained with coomassie......
sorry if you already know this!
i used 2 makers one stained (bio-rad) and one unstaind (amersham biosciences) they gave the same result.
As far as I know, the band size depends on the aa composition of your protein and isoelectric point and so on. Just to give you an example: the real size of Mdm2 is 55KDa but it runs as a 90KDa protein. You can check on th edatasheet of your Ab which size they expect. Then there is another possibility: if you are transfecting it and it has a tag, the tag could change its size
hi thanks,
1. i ran the proein on a SDS-PAGE gel (15%) and therefore i think its suppose to run only by it's size - am i wrong ?
2. i use a flag tag (7aa) and the 18kda is after taking that in consideration.
3. does anyone know why mdm2 runs as a 90kda ???
As far as I know, the band size depends on the aa composition of your protein and isoelectric point and so on. Just to give you an example: the real size of Mdm2 is 55KDa but it runs as a 90KDa protein. You can check on th edatasheet of your Ab which size they expect. Then there is another possibility: if you are transfecting it and it has a tag, the tag could change its size
hi thanks,
1. i ran the proein on a SDS-PAGE gel (15%) and therefore i think its suppose to run only by it's size - am i wrong ?
2. i use a flag tag (7aa) and the 18kda is after taking that in consideration.
3. does anyone know why mdm2 runs as a 90kda ???
Mdm2 had an apparent MW different from the real one because of its aminoacidic composition (very positively charged). Moreover:
"Often questions are posed regarding apparent discrepancies between protein size as determined
by gels vs. other methods, such as sequence analysis. Two factors explain most of the observed
variation.
The first factor is the amount of SDS bound to the protein. SDS is employed to disrupt secondary
structure and give all proteins a constant charge/ mass ratio, which is assumed to be 1.2g SDS/g
protein. However, as stated in a review by Hjelmeland and Chrambach1; 'this assumption fails
more frequently than is generally known.' The most common deviation from this assumption is
probably a lower than normal amount of bound SDS. All else being equal, mobility would
decrease, since the protein would have less of a negative charge.
A second source of error in molecular weight estimates, is that protein mobility in the gel is more
a function of molecular size (which is a function of both weight and length) than of molecular
mass. It's generally assumed that SDS proteins all exist in a random coil form, so the relationship
between length and mass should be constant. Even assuming constant charge, if a protein has
unreduced disulfide bonds or areas of incompletely disrupted secondary structure, it cannot
unfold to full length and, it would tend to run faster than expected in a typical SDS gel.
These deviations from the ideal can combine in every conceivable way, making it difficult to
predict a net effect on migration rate."
From: http://www.mnstate.edu/provost/SDSPAGE_MW_Estimation.pdf
If it is not enough, you can also have a look at this: http://www.invitrogen.co.jp/focus/201024.pdf
thank you !!!!
didn't know that !
So do u think i should look into post-modification options or just except the fact that the protein runs as a 25kDa ??
Lior.
didn't know that !
So do u think i should look into post-modification options or just except the fact that the protein runs as a 25kDa ??
Lior.
Can you express it in bacteria somehow? You could run the two samples (bacteria and cells expressed) and compare the mobility. If you see a difference, it could be because of ptm, otherwise it's just the way it runs. You could also try and buy it if it is available (for ex. Proteinone sells proteins).
[/quote]
Can you express it in bacteria somehow? You could run the two samples (bacteria and cells expressed) and compare the mobility. If you see a difference, it could be because of ptm, otherwise it's just the way it runs. You could also try and buy it if it is available (for ex. Proteinone sells proteins).
[/quote]
can i simply transfer the cDNA in to a bactria expression plamid ?
[quote name='Lior_m' date='Aug 2 2006, 01:30 PM' post='62234']
[/quote]
Can you express it in bacteria somehow? You could run the two samples (bacteria and cells expressed) and compare the mobility. If you see a difference, it could be because of ptm, otherwise it's just the way it runs. You could also try and buy it if it is available (for ex. Proteinone sells proteins).
[/quote]
can i simply transfer the cDNA in to a bactria expression plamid ?
[/quote]
Yes, you can