Hi to everyone, I am currently working on western blot for a lower Kda protein ranges around 30-50, i am using 70v and 90mins for transfer at room temperature but still the protein remains in gel itself, can anyone suggest me a solution for this? Buffer i used is Tris-glycine along with methanol and pH is 8.3. I have also done the ponceau to see is their any proteins in membrane, but i couldn't find any. i can see the proteins in my samples on gel using commassive but ladder were missing in gel as well as blot.
Hello Muthukumaran,
Since you see no proteins at all on the membrane after transfer, the first thing that comes to mind is reversed polarity - have you checked that you are transferring in the right direction? You may still have residual protein in the gel, that would be stained by Coomassie.
Best wishes,
Roland.
Hi, may be it is running out of the gel. Did you try 10 or 12% gel?
I routinely detect proteins in that range using either a 7.5% or 10% Criterion TGX gel transferred onto methanol activated PVDF membrane (Immobilon P) using ice-pack chilled 1x Biorad Tris-Glycine supplemented with 20% methanol. I use the Criterion transfer system set at 95V for ~1-2hrs.
I've tried cutting the [methanol] in the transfer buffer down to 5% or even 0% for such low-mass proteins, but it seems to reduce their adherence to the membrane. The proteins transfer out of the gel okay, but they do not seem to stick to the membrane with low [methanol]. The higher methanol seemed to fix this problem, but my experience is anecdotal at best.
You say that the protein is staying in the gel. Have you checked it, or is this just an assumption? You could check to see if you're getting poor transfer by coomassie staining the gel post-transfer. After the transfer you should see no ladder left in the gel, and there should be little to no protein left in the gel (as detected by coomassie stain). There may be a little bit of high molecular weight stuff left, but that's almost unavoidable. Silver stain could be used for the same purpose.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques