I had purified two isoenzymes with two different pI values which were 7.2 and 4.1 respectively. However, when I run activity staining on 12.5% native-PAGE, the band appeared for both isoenzymes were in close-position. Here I attach the gel picture for your reference. Is there any explanation on this problem or is it common to have this kind of result? Thank you
Hi there,
As far as I understand, the profile you show correspond to the resolution of the two isoenzymes on native gel, right? If yes, what is the problem you are talking about? You have in hands two different isoforms with different pI you eventually manage to resolve on native gel. It's just perfect, what were you expecting further? Native gel is not IEF where proteins are resolved only in function of their pI. On native gel, the protein mobility is function of the size and of the apparent charge of the protein at the work pH (which is not strictly correlated to pI value). So the only thing I can say about your gel profile is that the lower band isoform is less negatively charged that the upper isoform ...
Bear in mind that just a few amino acid substitutions on the surface of some proteins can significantly change their pI, so it is feasible for two isozymes to have similar apparent size, yet different pI. Human calmodulin (MW = 16.8k) has an average pI of 4.09. If you substitute 10 arg for 10 asp this changes to 5.27. If you tack on another 5 arg, it becomes 8. So for 10 substitutions and 5 additional a.a.s you get a similar change in pI to that in your isoenzymes for a theoretical MW change of just 1k.
Hi there,
As far as I understand, the profile you show correspond to the resolution of the two isoenzymes on native gel, right? If yes, what is the problem you are talking about? You have in hands two different isoforms with different pI you eventually manage to resolve on native gel. It's just perfect, what were you expecting further? Native gel is not IEF where proteins are resolved only in function of their pI. On native gel, the protein mobility is function of the size and of the apparent charge of the protein at the work pH (which is not strictly correlated to pI value). So the only thing I can say about your gel profile is that the lower band isoform is less negatively charged that the upper isoform ...
Hi Nik, I agree with Dominique in that native PAGE is dependent on size, shape and charge of your proteins. IEF only depends on the pI of your protein, Thus, native PAGE and IEF are not comparable.
Hi Nik, Im sorry if my answer not helping you much. Im not sure how far pI can affect the mobility of the protein in NATIVE-PAGE.
But, if you come back to basic, in native PAGE, proteins are separated according to THREE characteristic:
1-net charge
2-size
3-shape of their native structure.
Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers (Laemmli's system, pH=8.4).
The higher the negative charge density (more charges per molecule mass), the faster a protein will migrate.
At the same time, the frictional force of the gel matrix creates a sieving effect, retarding the movement of proteins according to their size and three-dimensional shape.
Small proteins face only a small frictional force while large proteins face a larger frictional force. Thus native PAGE separates proteins based upon both their charge and mass.
Both of your protein was acidic and become negatively charge in Laemmli's buffer system. and as other respondent saying, its not just charge of your protein, their mass also take part. For the conclusion, i quote the answer from Bevan Cumbes "it is feasible for two isozymes to have similar apparent size, yet different pI" because there are other factor that affect how the enzyme migrate in NATIVE-PAGE, not just only charge. Please do correct me if im wrong.
Sorry for the long answer.
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