Western blot transfer issues

Question

I am having some issues with transfer efficiency to nitrocellulose membranes. To get all the technical info out of the way:

Transfer method: Wet, tank transfer overnight at 30V with normal tris-glycine transfer buffer + 20% methanol. I do this at RT with stirring. Of note: I have not recently been equilibrating my gel in transfer buffer, as this was reported to decrease transfer of basic proteins due to the stripping of SDS (I can furnish this reference if anyone is interested).

Sample: In all cases these are in vitro reconstituted nucleosomes with or without certain enzymes--the main transfer issues are the core histones, which are highly basic (pI 10-11). There should be no more than about 100 ng of each histone being transferred per lane. The salt in the sample is <25 mM. They are boiled prior to loading. There is no noticeable precipitate in the samples before or after boiling, however the volume is quite small (20 ul) so I would not necessarily detect precipitation.

Nitrocellulose is 0.2 um and is pre-equilibrated in transfer buffer prior to transfer.

The gel: I use a 15% gel, 1.0-1.5 mm thick. The acrylamide I use is 37.5:1 acrylamide:bis. Some issues could arise here--the acrylamide solution is about 1 year old and has been stored at RT that entire time (which is what is recommended on the label). In addition, we typically keep a stock of 10% APS at 4C, so I cannot guarantee my APS has not gone bad.

I have been quite careful generally to ensure there are no bubbles between any components of the sandwich and filter paper/foam pads are soaked in transfer buffer.

Now to the issue. Attached are images of the membrane after transfer (and also after processing, thus the relatively high background from the blocking buffer) as well as the gel after transfer. As you can see from lane to lane I seem to have differing transfer efficiencies. This has occurred with my last three blots. My first thought was sample precipitation, but I have not had this issue before despite running essentially the same blot many many times over the past year.

Any thoughts? Quite frustrating, as it has appeared suddenly with identical protocols/reagents as before.

Answer 1

The first thing that comes to mind for me is that the transfer seems excessive... isn't histone sub-20 kDa? I have often transferred similar-sized proteins at 4C for 30 min, 30V.

In general, presence of SDS will inhibit transfer efficiency, and increasing alcohol in your TG buffer will counteract the effect of SDS present. Perhaps bring down the methanol to 10%? I would be curious to see that reference regarding the SDS/pre-equilibration, though. That certainly runs contrary to how I understand it working. (Note: I flip-flop between equilibrating or not equilibrated my gels without any observable differences.)

If your acrylamide or ACS had gone bad, your gel wouldn't set.

If you don't already, I'd suggest running a Bradford or BCA on your samples to be sure that you're loading the same amount into each well. Having loaded more into each well could give the appearance of uneven transfer.

To be honest, I don't really see what you mean by uneven transfer, based off the Coomassie and Ponceau. If you're seeing a gradient effect across a large portion of the gel, I would suggest you visually inspect to make sure that the gels you cast are even width all the way across. Having a thicker gel in one place than in another could impact the resistance (heat) in that part of the gel and impact transfer. If you're seeing a lane-by-lane transfer efficiency shift, I would suggest localized pH or temperature changes due to uneven loading of salts, protein concentration, or lipid concentration.

Hope this helps!