Native PAGE - (Oct/24/2011 )

Hi everyone,

Is there anyone running native PAGE for protein analysis?

I met some problems when using this kind of protein electrophoresis to separate protein bands. It's because my protein is positively charged. So I'm thinking about increasing pH of buffers (including sample buffer, running buffer and gel buffer): pH >pI. So that my protein could be negatively charged.

But I've not tried but I'm not sure about this solution.

Anyone can help me? Thank you so much!

You must be very careful when doing this, particularly if you are using a stacking gel. Many people think that the stacking gel stacks because of a difference in percentage acrylamide, but it's actually a difference in ionization of glycine (in Tris-glycine gels) creating a charge gap. Obviously, different gel systems work differently (i.e. Tris-Tricene, Bis-Tris, etc), but as a general rule with native PAGE, you want to stay away from pH extremes because your proteins will start to denature (which will smear your bands) and the electronic properties of your gel/running buffer system will be distorted, causing very weird gel artifacts.

If you are buying your gels/gel system from a company, try contacting their tech support to see if they have any recommendations.

Best of Luck.

Thanks for your advice

Because this is the first time I work with protein, I actually don't have any experience in this situation. However, I used continuous gel (15%). Also the pI of my protein is about 8.83. Thus I just want to try once more with the pH 9.0 or 8.9.

I run at pH 8.8 and 8.3 last time (with the same steps) but I couldn't see any band. So I guess that my protein didn't move in the gel.

By the way, I tried to switch electrodes but it didn't work successfully.

Try blue-native gel???

Do you think it worked in case of my protein?

The coomassie brilliant blue G in BN-PAGE binds to proteins and confers a negative charge without denaturing the protein.
Blue native page separate the protein by size (run through a gradient page-gel).

I not sure whether it worked for your protein but you can try and find out.

Thank you so much for sharing this I will try and hope that it'll work efficiently

The BN-PAGE is a good idea, but with a pI=8.8, you might just try dropping your pH to 6.8-7.2 and reversing the field polarity. At a pH range of 8.3-9.0, you will have very little protein that is charged, and what is charged will be very minimally charged. Either way, it's worth trying, as long as you have enough sample to work with.

Best of Luck.

When I purified this protein, I eluted it with a buffer containing high concentration of Urea (8M). And I wonder that if it has effect on protein electrophoresis?

annie.moore on Tue Oct 25 03:11:19 2011 said:

is it still in urea when you apply to the gel? urea denatures (somewhat reversibly) the protein.

urea in the gel enhances charge separation.

since your pI is so high then a neutral or acid pH gel (with reversed electrodes, + to -) should be used for separation.

just reversing the electrodes when the pH is so near the pI will make virtually no difference since charge on the protein will be minimal if at all.