Tricine - Fixation of protein (Dec/26/2008 )

Dear All,
I have been working with 1.1 kDa protein (approximately). Use Tricine electrophoresis for my proteins. Fix the protein with gluteraldehyde. Is there any other method for fixing my protein as it takes long time to wash my gel to remove the gluteraldehyde and if any trace is present, staining problem occurs.

I do not get any band in my gel. I load 20muL sample (inclusive of Sample Buffer). I use 22x15 gel for my separation of protein from other.

I also get a layer at the bottom, below the dye front region ( dye front is not visible) after staining.

Please help me in the aspect.

-Vidhyasagar-

we use methanol/acetic acid (50%/12%) to fix tricine gels before silver staining.

we fix with 50%/7% 0.25% coomassie blue r-250 then destain with 30%/7% for cbb staining.

-mdfenko-

QUOTE (mdfenko @ Dec 29 2008, 10:33 AM)

we use methanol/acetic acid (50%/12%) to fix tricine gels before silver staining.

we fix with 50%/7% 0.25% coomassie blue r-250 then destain with 30%/7% for cbb staining.

We tried this fixative but our protein gets diffused and is not properly fixed in the gel. Hence we opt for gluteraldehyde fixative as given in Sigma instruction. But we face problem of removing the gluteraldehyde from the gel.

We also now face another problem of smiling of protein band in gel. Is there any reason fro this

-Vidhyasagar-

we have looked at enkephalins (500-600Da) on these gels and have not suffered losses from diffusion.

what is the acrylamide percentage of your gel?

we use 16.5% or 10-20% (sharpens the banding) gradient acrylamide for low mw peptides.

smiling usually occurs due to uneven heating of the gel (warmer in the middle than at the sides). you can either cool the gel or reduce the power when running.

-mdfenko-

Thank You. I will try with 18% gel. Will try with Ur suggestions

-Vidhyasagar-