Hello,
I'm having trouble with my western blot transfer using Biorad Trans-Blot semi-dry system. It seems that very little protein gets transferred, and the rest is left on the gel. I did Ponceau stain and saw a few bands on my PVDF membrane, and after a Coomassie stain of the gel (after transfer), there were still multiple bands, especially the high molecular weight ones left on the gel. Please see attached images of the Ponceau stain (with ladder), and the Commassie stained gel. The ladder transferred just fine, which is why I didn't suspect anything until I did Coomassie. Is there a good explanation why the ladder (protein standard) transferred ok but everything else didn't?
I use the High MW transfer protocol (2.5A, 25V, 10min). This is the highest volt and amp the manual recommended. Would a longer transfer work?
The only thing I could think of that might caused this issue is that I use NuPage Bis-Tris gel (not Biorad) running in NuPage buffer (MOPS-SDS) and then transferred using Biorad Turbo Transfer pack, and their proprietary buffers might not be compatible(?), although I rinsed the gel after it finished running so any SDS in the buffer should not carry over... Not sure if the is a real issue though.
Any help would be greatly appreciated. Thanks!
Pawat
I would not "mix and match" different types of buffers, gels etc from different vendors.
In order to ensure reproducibilty and optimal results, companies sometimes design their equipment to be compatible with few reagents.
I have previously used this equipment to transfer protein using TGX Fast Cast hand cast gels (BioRad) and hand-made transfer casettes (blot paper + PVDF membrane) using Lamelli buffer. BioRad does provide a formula for lab-made transfer buffer.
Is the green ladder lowest mW protein? It looks like you are actually transferring the lower mW proteins as seen by the Ponceau stain but not the higher mW protein as evidenced by the coomassie. I am not a big fan of using the ladder as an indicator of transfer as I have had experiences where the ladder did not provide a good correlation of transfer. Also note that higher mW proteins transfer slower and last, leading to the undertransfer phenomena, whilst lower mW proteins transfer faster leading to the overtransfer phenomena. The transfer of high mW proteins has been a problem in the past, I have heard people using overnight protocols in fridges/cold rooms to transfer these proteins using low voltage settings. Your problem may be that you are using a different type of gel (and perhaps buffers) that is not optimized to the conditons of the device. Variables such as gel thickness (i.e. 1mm vs 1.5) have been known to make differences in transfer behaviour.
As Can says, once you are into proprietary reagent systems its usually best to adhere to them completely, until you can swap out individual components and see if the system remains functional. Much of commercial product development goes toward locking the customer into an entire system of consumables as well as limiting shelf life to assure regular repurchase of components. Some of the patents (or even SDSs) can allow the user to figure out the magic components (as in many Qiagen products) but companies make a real effort to avoid users "DIYing" their products, or allowing mixing and matching. (Its also the case that sometimes all the components are simply required without any conspiratorial efforts!)
There is no downside to doing multiple transfer cycles on these (high current, low voltage) transfer units, as long as they don't dry out and cook your gel; I'd try running >2 cycles in a cold room with 2-3 membranes to control for bleed through on the low MW proteins.
Ahh.. Thanks y'all. Just got Biorad gels and looks like it transferred much better this time. Still some high MW proteins left on the Coomassie stained gel but mostly everything got transferred.
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