I need some help making/running Native PAGE - (Apr/11/2008 )

I need some help on trying to run a Native PAGE:

I am trying to separate a protein of interest from cell extracts. The protein is 62kDA, with a pI=8.74.

I tried running it on a resolving gel that is 7.5% (29:1 polyacrylamide:bis) (with a 5.2% stacking gel) but leaving out the SDS.

Anode Chamber buffer is 25 mM Tris and 192 mM glycine (pH8.8) and Cathode Chamber buffer is the same but with 1% DOC

Gel was pre-run for 30 minutes at 40mA, and then electrophoresis was carried out for 1 hr at 60mA.

Resolving gel pH was 8.8
Stacking gel pH was 6.8
Running buffer pH was 8.8

After separation, I place the gel in 1X SDS running buffer for 5 minutes. Then I transfer on to nitrocellulose for 1hr at 117V and finish immunoblotting.

It looks like my specific bands are going through the stacking gel but stop at the top of the interface. I am not sure but I think it's due to the rise in pH in the resolving gel which slightly higher than the pI of my protein of interest. It may also be do to the higher percentage acrylamide of the resolving gel.

I have also tried using 37.5:1 (polyacrylamide:bis) but not much different results. I will next try using the pH 6.8 buffer in both the stacking and resolving gels and also use a lower pH running buffer (maybe 6.8 as well).....

If you can help, PLEASE DO SO!!!!!!!

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if you use pH 6.8 then you will have to reverse the electrodes.

the pI of your protein is high. you may want to try an acid gel (you can find the protocol in the forum).

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you said that I would have to reverse the electrodes; but when I ran it before using a pH 8.5 buffer, it went fine through the stacking gel (pH 6.8) but stopped at the resolving gel (pH 8.8)

Should I just make both stacking and resolving gels at (pH 6.8) and leave the running buffer at pH 8.5? I am a little weary of reversing electrodes, I am afraid that my protein may go out of the wells. Sorry if this seems dumb, but chemistry is not my strength.

Thanks for the help mdfenko

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the pI of your protein is higher than the pH of your buffer so the protein will carry a positive charge. your pH 8.5 buffer is very close to the pI (and may actually be higher if it is a little off) so the protein may have migrated a little.

but 6.8 is significantly lower than the pI so your protein will carry a stronger positive charge. if you run the gel at pH 6.8 then you will need to reverse your electrodes so that you run from positive to negative.

you could switch to ornstein and davis chemistry (you can also find this in the forum). it runs at pH 9.5 so you can run with normal electrodes but your protein will run more slowly than you may want.

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You`d better try to use Blue-native Gel , any question ,contact with me

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http://search.vadlo.com/b/q?p=2&sn=158...rel=0&srt=0

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