Is there any way to do efficient transfer from 15% SDS PAGE gel for proteins of 14/16 kDa in size AND 62 kDa in size. I'm using wet tank transfer method and Immobilon-FL membrane (PVDF). My transfer buffer is 25 mM Tris, 192 mM glycine, 20% methanol. Usually I do 200 mA for 2 h. The problem is that quite often I see some of the standard bands (at least 75 kDa and bigger) still on the gel. With 8% gel these transfer conditions are still okay. I am not sure which conditions I should optimize.
For a 14 KDa protein, if think you could run a gel at 12% without any problem.
In addition, when I stain a gel with a colloidal blue, after the transfer, I could see some proteins at an "high" molecular weight but even at a lower mass. In all case, it was not a real problem to see "my" protein in western blotting. The transfert is not a very efficient technology....
In my point of view, try to do your western and look if you see your protein.
If you see nothing, you could try of gradient gel between 8 to 15% of acrylamid.
Sorry, I forgot to mention that my antibody recognize both 14 and 16 kDa bands and I have to be able to separate those two bands. I think 12% is not enough for this purpose. I have always proceed to antibody incubation but I'm a bit worried whether that will give proper results if part of the 50kDa and 75kDa ladders are still on the gel... Because I would like to get quantitative results.
I think that the transfer of your protein at 62KDa will be "quantitative" even if its transfer is not total.
If you want to obtain an homogenous transfer along the gel, do a gradient.
Try with 350mA for 90 min. After 60 min change the ice (or the blue cooling unit) in the wet transfer tank with a new unit and proceed for other 30 min .
I usually transfer proteins from 15% gel to both PVDF or nitrocellulose membrane with this method and it works.
Hi Sonja. Generally, the higher the molecular weight of the proteins, the more difficult it transfers from the gel to the membrane. And since you are using a 15% gel, this might also make it a little bit more difficult for the larger proteins. However, this shouldn't be too big of a problem. It can be quite difficult to transfer everything of "large protein" but like Stephane said, even if everything doesn't transfer, you should still be able to quantify. If you're only interested in the 14kDa, 16kDa, and 62kDa bands, and you can pick up nice strong bands after immunodetection with those transfer conditions, I would suggest that you stick with it. Otherwise, you could increase the transfer time a bit to allow more of the 62kDa protein to transfer, but just make sure that the smaller proteins are not transferring through the membrane then (use two membranes). Or you could decrease the methanol to 10%. Otherwise, you could also use the Immobilon membrane designed for smaller proteins, think its called Immobilon-PSQ, it might give you some flexibility with optimizing for the larger protein while keeping the smaller proteins on the membrane.
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