incomplete transfer of protein samples for western blot

[curious1]

Hi Everyone,

I recently starting having trouble getting complete transfers on my western blots. I transfer at 1 hour at room temperature at 100V and it has always worked fine. I made fresh transfer buffer and tried transferring overnight, even used a new power source and still the proteins don't transfer over or very lightly. I once tried transferring at RT for 1 hr at 100V, then O/N at 4 degrees at the lowest voltage setting, and then increasing the voltage to 100V for one hour at 4 degree and that helped, but there has to be another way! It shouldn't take this long to transfer and not even completely either! Anyone have any ideas?

[blcr11]

It sounds like either you're not getting enough pressure on the membrane sandwich, so that the contact between the gel and the membrane is flaky, and/or you've got a current leak bypassing the gel/membrane sandwich. Try adding another gauze pad or two (or whatever you're using as spacers) or replace all your spacers with fresh ones. They get squished (at least mine do) with use and don't fill up the open space so well. Look around and make sure all the wires are connected tightly and that they aren't significantly corroded. If they are, you might want to consider replacing the chamber. Also look for any cracks or holes in the plastic that could be the source of a current leak. You've made fresh buffer and I doubt you would make a mistake there more than once, but you could change it anyway, just in case. I presume the current is what you expect. Starting out it's not almost zero by any chance, is it?--if it is, that's why the transfer is poor, but it doesn't explain why the current is too low.

[blcr11]

I forgot toe add--you can overdo it, too. If you run too long, you can run your proteins right through the membrane and off into solution. I kind of doubt that's what's happening here.

[curious1]

Ah, it turns out it was a loose wire. I've connected the wire back and it's running, but the mAmps seem a bit high, around 400mAmps. Is that going to be a problem?

[blcr11]

That depends on how large your gel system is and the specifics of the transfer buffer you use. It could be normal, I don't know. It's high for the system I'm used to (Novex miniblot) which runs between 30-60 mA to start. The only danger from too much current--beyond heating, anyway--is running the lower MW proteins clear through the membrane. I would just try a run of standards with the usual 1 hour transfer protocol and see how it looks. My guess is you're probably OK.

[curious1]

hi blcr11,

Thanks for your reply! hmmm, the blot looks OK, the ladder transfers over just fine but since I am trying to detect a very small MW protein (~18kd) I do worry about the proteins tranferring right through. Standard protocols tell me to run at 100V which is what I do, but the current is always so high. Should I decrease the voltage then? Or just run at 200mAmps?

[blcr11]

Not sure what to tell you. Most likely you're OK running at the higher current. For a one hour run, even at higher current, the risk of running an 18kD protein through the membrane is probably not that great. I use pre-stained markers and you can see that they don't move all the way through, but are retained on the "gel' side of the membrane.