Hi, we tried to transfer proteins from an acrylamide gel 1.5mm made with our recipe to a PVDF membrane of BioRad in the Transblot Turbo of BioRad, but the majority of protein rested in the gel. Could it be a problem of the thickness of the gel or due to the recipe used? We like to know what kind of recipe do you use to made the gels and which protocols do you follow.
I've found that the Transblot Turbo does not transfer so well with made gels (I was making 12% stacking gels 1 mm thick ). At least not to NCF. Have not tried PVDF. I was getting completely different results when comparing made gels to the biorad TGX gels. So were others in the lab. Because of this reason we switched to only using the Biorad TGX 4-20% gels and transferring for 7 min.
I did transfer for 20 minutes for a 38 kDa protein and it did not transfer correctly.
We have 1.5mm homemade acrylamide gels and with both 7.5% and 15% gels we did not get good transfers with the turbo to PVDF membranes. Attached is an image of a coomassie stain of 2 (7.5%) gels AFTER TRANSFER using both methods:
Top:Wet transfer 1hr, 100V, our own Towbin buffer recipe
Bottom:Transblot turbo (10 min, 1.3a, 25V) with Wypall stacks & BioRad towbin buffer
As you can see, MUCH more protein was transferred using the wet method.
We get even less transfer, and it's very patchy when we use 15% gels, so we have gone back to using the 1hr wet transfer method.
transferring 1.5mm homemade gels with the transblot turbo is definently a bit problematic. We get good transfer with homemade 1mm gels using the transblot turbo but the 1.5mm transfer is proving frustratingly difficult. We transferred for 25mins at 1.5A and the transfer onto nitrocellulose was not great quite poor actually. Will need to try and transfer for longer and see how that works
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