hi

1-use fresh Bis-acrylamide / APS & TEMED If possible .

2- check miliAmp during transfer ( constant and right value mA).

3- wait for complete gel formation ( instead of DDW you can used isoperapanol on gel) dont hurry . gel formation induce in 37 c .

4- check pH of transfer buffer and decrese voltage ( such as 80 V for 80 min )

5- you can used PVDF (0.2 um)

6-SDS page Run on Ice tray or cold room.

7- cut staking layer before Blotting .

good luck

Hello Reza,

thank you for your answer!

The suggestions 1, 3, 6 and 7 would certainly not account for my problem. Using semi-dry blot does not give me the problem. Interestingly a colleague of mine looked at coomassie stained gels after blotting and saw the artifacts in the same way in the residual proteins in the gel. I think the gels themselves are fine. It must be a transfer problem.

4- I didn't check pH of transfer buffer yet. I decreased the voltage already, maybe I am somehow blotting too long? mA during transfer usually is about 120 mA. I should check more carefully.

5 - PVDF usage doesn't change anything. I checked already.

Dear Chris,

I faced not the same but similar problems with my blots. Event after following the same protocol for years suddenly start facing undetectable problems. And one start questioning himself, which is frustrating and takeoff your central research focus. I will just buy readymade “NuPAGE® Protein Gels” for your precious samples and do the experiments. Best Faiz  

Thank you Faiz. But again: It is not a gel problem, but a transfer problem.

Hi Christian

- hast Du mal die Gelkammer geprüft?? Möglicherweise gibt es einen

Elektoden-Defekt??? Grüsse, Petra

Hallo Petra!

Danke für den Vorschlag. Die Elektroden sehen einwandfrei aus und ich sehe auch keinen unüblichen Stromfluss oder einen Unterschied zwischen verschiedenen Kammern, wenn ich diese Parallel laufen lasse. Aber das ist natürlich ein guter Punkt, weshalb ich noch einmal genau drauf geschaut habe. Die Kammer-Einsätze sind vollkommen unbeschadet.

Looks like there are more problems with this blot than just the transfer hole pattern, but to address this latter issue, the only similar thing I've seen (not identical) was traced to protein contaminated transfer sponges. The sponges had been stored in dH2O and grew bacteria during storage! Try replacing them with new ones, or wash them in hot 1 M NaOH and rinse very thoroughly in water, then store in transfer buffer for at least 1 h prior to use. No guarantees, but it worked for us.

Thank you, Norbert!

You are of course right, that the example above looks quite bad. Obviously, there is a rim, where the membrane has dried out or something. However, what you see is an OxiBlot. So in principle, it es expected to see smears over the whole membrane. But you could never compare the intensities between lanes with such an artifact and also this was the first shot. So yes, results should look better!

Attached here you see a coomassie staining of residiual protein in the gel after the wetblot transfer. Same picture with these holes, indicating that the proteins move out of the gel faster where the holes are. I still don't see how this happens in a tank full of buffer...

We boiled the sponges and washed them intensively. It doesn't help. If it was the sponges, then I would expect the opposite problem (dark spots where the holes are), which most people find to come from some dirt or bacteria in the sponges. So I think, it is not the sponges...

*sigh*  :-(

I sympathize with your frustration, Christian. You know there is no magic involved here. You got good results before, so there is some systematic error involved, and there is no reason why you can't get good results again. When this happens (and I have seen it many times in many unrelated procedures) the best solution is usually to start fresh. Throw out all your buffers and reagents and get someone in another lab (who's competence is trusted) to make new reagents, from chemical stocks that are trusted and not the ones that you use  in your own lab. I say this because we are creatures of habit, and we make systematic mistakes that we are unable to see objectively at times. If this remake of reagents is done from accurate recipes by competent people and you run your gels (preferably on someone else's trusted equipment) you know you MUST get good results again. Then comes the tedious task of replacing one item after another with your own to find out where the problem is. At this point, in my experience, often things just continue to work and what was actually wrong remains a mystery that is eventually forgotten... until the problem recurs years later! Good luck!

Thanks again. I am sure, there is some systematic problem in our lab. We're going through the one by one replacement strategy for some time now and still didn't find the solution. (We exchanged blotting chambers, power-supplies, sponges, filter papers, made all buffers new, but not yet the stock chemicals, etc...)  Even though we use the same membranes for years, I will as next get some membranes from other labs to see whether something is wrong about our membranes.

I know that is the kind of frustration, that one has to cope with in science. Sooner or later we'll solve the problem, knowing what it was or not. Hope I can respond to you all with problem solved message soon!

Good luck with all your own work and thanks a lot for taking the time to discuss our tech problems with me! I really appreciate that!

Update:

After I had gone through the one-by-one-replacement strategy for every idea I could come up with concerning the blotting procedure (without any success finding out, what the problem is), I decided to go the hard way now.

I exchanged EVERY solution, chemical or whatsoever included in the whole process including sample prep and SDS-page. Tris-Glycin-Buffers, Lämmli buffer, SDS, lysis buffer, ponceau, running buffers, blotting buffers, temed, acrylamid bottle, membranes, whatman papers... whatever I found to be used.

After five absolutely successful artifact-free wetblot-transfers and very nice ponceau-stainings as I like it, maybe I can carefully tell: Problem solved. No idea, what was the reason, but hopefully I'll never have to search for the reason again.

Thanks again for all your input and best wishes.

Chris