native gel problem - (Feb/15/2008 )

Hello,
Can someone help me? I am pretty exhausted with the native gels. I have tried to run a native gel for the protein that is 18 kDa and has pI of 5.54. I have used 12% separating gel ph 8.8 and 3% stacking gel pH 6.8. Running buffer pH is 8,6. After running the gel for 30 min or 2h at 170 V I can see nothing in my gel. So does my protein move at all?

Sarita

Hello,

thanks for reply. I dont use ladders since in native gels I cannot predict the size. But when I stain the crude protein samples with coomassie I can see the proteins. I looked at my gels more closely now and I can see that my protein is in the between the stacking gel and resolving gel. So the protein do not enter to the resolving gel. Perhaps the resolving gel is too tight? Maybe I should change my resolving gel from 12% to 7% ? I could also just run longer time but Im afraid that the gels may heat to much and denaturate my protein. I need the protein in active form to perform activity tests.

do you know if your protein could dimerize with something else?

I dont know about the dimerization.

try a gradient gel, 5-15% should allow your protein to enter and migrate.

and you will be able to enhance size separation so you can try a ladder (if not already in sds and reducing agent) or you can load some known proteins (make your own ladder).

but missele is probably correct, your protein is probably aggregating.

Are you certain of the pI? Is this calculated or observed?

The pI is calculated based on predicted amino acid sequence; the gene has been recently cloned.

It is also possible that the protein is dimer in native form and it can be either homodimer or heterodimer. I am running crude protein samples extracted from plants.

I used 10% resolving gel and the protein still did not enter to the gel. After running 7.5% resolving gel I could not find the protein from the gel anymoro.