what are the best transfer conditions? - (Feb/26/2012 )
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
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.com/immunodetection/id3/membraneselection). 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.
Sorry. I forgot to include the transfer conditions.
We transfer at 150 V for about 1 hour, using PVDF membrane.
Tris-glycine buffer is very common for wet transfers. Usually this is called Towbin's buffer when methanol is added, is is often very similar in composition to running buffer. The methanol is not essential, but helps strip the SDS from the protein, and increase binding of small proteins to the membrane. Addition of SDS can increase the solubility of the proteins such that they are transferred effectively.
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.
The above mentioned conditions (
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.