protein travels differently under different SDS-Page conditions - (Jan/08/2008 )
Hey,
I'm working on a poorly described mitochondrial protein. It becomes imported into mitochondria and afterwards processed. Thus, I can detect two bands: A premature and a mature form. I happen to have a very good antibody that gives me absolutely no backround but detects specifically these two bands. The company who produced it raised it against the full length peptide and tested it only by doing Western Blot against this peptide. But I did an in vitro mitochondrial import assay to be sure that the second band is indeed a result from processing the premature protein. All great.
However, when I run the same samples under different SDS-Page conditions I get different sizes for the mature protein. It appears about 5kDa smaller when Tris-HCl or Bis-Tris Gels with Laemmli buffer and MOPS running buffer is used than when I use Tris-Tricine with Tris-Tricine loading buffer and Tris-Tricine running buffer.
Does any of you smart guys out there have an explanation for this? I know that the predicted (all informations for this protein are only predictions...) isoelectric point of this protein is very high with 9.9845 but can that have such a huge influence on traveling under different conditions?
I really appreciate any ideas that help me to understand what's going on.
Thanks a lot in advance,
Henrike
Does any of you smart guys out there have an explanation for this? I know that the predicted (all informations for this protein are only predictions...) isoelectric point of this protein is very high with 9.9845 but can that have such a huge influence on traveling under different conditions?
in sds the pI is a lot less important. the sds imparts a net negative charge to the protein.
tricine is used primarily for the separation of small proteins and peptides (<20kDa), you will get more accurate determination of the mw of a small protein. what is the mw of your protein? is 5kDa significant?
sds page gives an estimate of mw. there are a number of factors that may affect the migration of a protein.
It's known that proteins migrate differently depending on the conditions of migration.
If you have a look on the datasheet of the protein weight markers you will see that they give you the relative weight depending on the gel which was used (tris glycine vs tris tricine)
Thanks guys for your responses.
The premature protein size is 24kDa and the mature form appears on Tris-Tricine Gel as 18kDa and on Tris-HCl about 13kDa. Since there is a predicted cleavage site for the mitochondrial import sequence that would result in a 18kDa mature protein I believe that this is the correct size.
I'm aware of the phenomenon that proteins migrate differently depending on the conditions of migration. It just puzzles me that this mature form migrates so differently while the premature form seems to be unchanged in it's migration behavior although the difference in size of the premature and the mature form is not that great at all.
I should add that I used 16% Tris-Tricine gels and only 12% Tris-HCl gels. Could that explain the differences in migration behavior?
Can I just assume that the 16% Tris-Tricine gel gives me a much more accurate information about the mature protein size and that when using 12% Tris-HCl the separation in this molecular weight range is not as precise?
I'm already about to do Mass Spec after running SDS-page under these two different conditions just because this drives me nuts... But I would rather like to spare me that extra work and move on. I'm mad about myself that I started using 12% Tris-HCl gels anyway. I guess I was just running out of precast 16% Tris-Tricine gels and was too lazy to cast my own...
Again, all your thoughts are highly appreciated!
Henrike
tris-tricine is more accurate than tris-hcl or tris-glycine when running small proteins and peptides.