you can look into this 8% non-denaturing PAGE. Lane 1: encircled : Dimer of 42 kDa. (A) Lane 2,3 and 6: encircled: 68 kDa (B) Lane 4 and 5 : mixture of both A and B , incubated to see their interaction. In rest of the lanes : loading dye alone. Protein A have pI = 4.5 and protein B have pI= 7.1. I use tris-glycine running buffer of pH=8.9.
It is possible for proteins to migrate out of molecular weight order on native PAGE, which separates on the basis of size, shape and charge.
I think it is possible, since you were using non-denaturing PAGE which separate protein in regards of their MW and net charge. If the bigger MW protein has more negative net charge than a smaller protein then it is possible to observe it migrate further.
Non-denaturing gels are tricky as MW goes. In such gels running distance has to do not only with protein size but also with charge (depending on the setting of the electrodes negatively charged proteins will run further down), how dense the protein are packed (SDS both charge and spread open the proteins), and interaction with other proteins.
All that said, I find the very faint bands hard to count on. Did you load very different amount of the 42 and the 68kDa and if so why? Or are they being stained very differently (suggesting very different pI)?
Yoram
You should repeat the native PAGE along with NativeMark protein mol. wt standards. If you do not have access to that, use, BSA, Ovalbumin and Cytochrome c in 3 separate lanes. This will give you an idea about at what size your own proteins are running. In case your protein in Lane 2 corresponds to its expected running size i.e around 68 KDa (this you can easily be compared to the lowest band (monomerc) of BSA), it will prove that your other protein (Lane 1) is running at much higher size, that indicates to me it probably exists in any higher oligomeric state (like tetramer or higher), that is very much possible. In other case if your protein in lane one runs at correct size as compared to marker, it will mean your other protein is degraded, that you can easily check, by taking part of your Native PAGE sample and add LDS loading buffer to it, and check it on SDS-PAGE. I hope this info proves useful to u.
Syed
Freiburg
A question ,are your proteins extraxcted from cell membrabne, or are cytoplasmic soluble ones? Such protein did not often migrate at the expected zone. What does it happen in SDS-PAGE
I am under the impression that Protein B is either not significantly entering the gel or very low in amount loaded. Very slow migration of Protein B may not be surprising if it has a pI of 7.1. As many people have been mentioning, migration in native gels is dependent on charge (measured by pI of the protein (pH at which the protein has a net charge of 0)) as well as Molecular Weight (MW). So, the pI of 7.1 for Protein B shows it to contain MUCH less groups that can be negatively charged than Protein A with pI of 4.5. The bands that you circle for Protein B are pretty faint. So, I repeat my suggestion that either very little of Protein B has been loaded (giving faint stainability) or the stained material that can be seen at the top of the stacking gel represents Protein B not significantly entering the gel due to it's low amount of negative charge. One way of proving if the migration of Protein B is due to low negative charge would be to run another gel at acidic pH (around 4 or lower) to see if Protein B now runs into the gel. If you do this, don't forget to switch the electrical poles so that the negative pole is at the bottom of the gel. One difficulty with seeing Protein B electrophoretic migration is that it's pI is near neutral. Interestingly, Protein B may not migrate well towards either the positive or negative poles if the pI of 7.1 is resulting from there not being many groups on the Protein that may potentially have either a negative or a positive charge (i.e. Protein B is somewhat hydrophobic). Increasing the concentration of Protein B (maybe 10 fold if you can) will show if you haven't loaded enough to truly see how Protein B is migrating.
Of course size is important, but charge is leading the movement of proteins in non denaturating PAGE. Yes it is possible what you found. Considering their pI, both proteins have negative charge at pH 8.9, bigger migration bigger negative charge, but as mention before there is no correspondence with pI of A and B.
Also, You shoud repeat the assay, using the same ammount of protein of A anb B
You can check that, running a Gradient Native PAGE (4-20% PAA) in order to separated them (A and B) by size, not by charge.
Hector Nolasco
CIBNOR
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