what are the best transfer conditions?
Posted 26 February 2012 - 09:34 PM
I have recently shifted from semidry to wet technique of immunoblotting which I have not used before. the transfer buffer used is composed of TRIS, glycine and 20% methanol.
My target proteins are as follows:
1) 105 kDa separated on 10% and sometimes 8% SDS gel
2) a number of proteins ranging from 12-35 kDa separated on 12% SDS gel
3) a number of proteins ranging from 40-60 kDa separated on 10% SDS gel
what would be an optimal constant current (or voltage) and transfer time for each category of proteins?
thanks a lot
Posted 27 February 2012 - 07:48 AM
My lab uses the Bio-Rad SDS-Page system to do wet transfers all the time. Your buffer formulation looks correct. We have no problem transferring the proteins in conditions 1 and 3 of your note, but my colleague had lots of problems transferring a protein in the 10 kDa range, close to your condition 2. I don't think he solved the problem. Sorry.
You didn't mention what membrane you use. We typically use PVDF (Immobilon-P) for our transfers. You could try the Immobilon-P(SQ) membrane, which has a tighter mesh for your condition 2. Here's a webpage to look at (http://www.millipore...mbraneselection). I'm sure Millipore would forward you a sample to try before buying a whole pack.
As I said, we use the whole Bio-Rad system and they may be able offer some suggestions for your condition 2 transfer (www.bio-rad.com).
I've attached the Bio-Rad Mini Trans-Blot manual. It might have some helpful tips.
Posted 27 February 2012 - 07:50 AM
We transfer at 150 V for about 1 hour, using PVDF membrane.
Posted 27 February 2012 - 01:00 PM
Small proteins are quite difficult to blot as they tend to pass through the membrane, you will probably have to play around with the time and voltage for yourself, but I would start with 75 volts for 30 min and work up from there.
150 v for 1 hour is far too much for proteins below about 50 kDa, 100 V for 1 hour will be heaps. For large proteins low and slow is best. I often transfer overnight at 15-30 V for pRB (105 kDa), though 4 h at 75 V works well.
Incidentally, 10% will not resolve proteins bigger than about 80 kDa well. You should be running gels of between 6 and 7.5% ideally.
Posted 29 February 2012 - 01:09 AM
in semidry blotting, I used to set the conditions to a constant current of 170mA/PVDF membrane, and if want to prepare 2 membranes the value of the current would be doubled to 340mA/2 PVDF membranes.