Semi dry transfer - Influence of isoelectric point on transfer (Dec/10/2004 )

Hi,

I'm using BioRad semi dry transfer following SDS-PAGE. Usually I run the transfer for 55 minutes at 15V. I've had difficulty detecting my protein of interest, CHK2 (65 kDa).

So I did a little checking and I found that the isoelectric point is 5.6 for CHK2. Would that influence the transfer process? My transfer buffer has a pH of 8.3.

Any ideas?

Thanks

Hi,

One more point I should add regarding the above post involving iso-electric point and protein transfer. I'm transfering to a PVDF membrane.

Hope this helps.

Have you stained the remaining gel with comassie blue to assure total protein have been transferred?
Voltage and time of transference depends not only on size of protein but gel thickness too. 60 V for 60 min (4 ºC) transfer proteins until 200 kDa in a 0,75 mm gel thickness. If you use 1,5 mm of gel thickness, try with 100 V for 60 min (at 4 ºC) or overnight at 15 V.

Hi,

In SDS buffers your protein coated by SDS molecules, so, carry negative charge and in most case should moving from negative pole to positive. And mobility will depend just on size of your protein. You can include on your gel pre-stained protein marker and its give you idea how your transfer good. If you can not see any bands in your gel after transfer, but all bands are on the membrane, your transfer is fine. If some bands still in your gel, I would increase duration of the transfer.

Sincerely,


Alexei