I run gradient gel (4%-12%), and do tank transfer. The transfer conditions however are different; 90 mAmp at 4degrees o/n. In the beginning, the voltage is less in the beginning, and by the end it rises but never more than 30V. I never experienced crumbling or expansion.

Hi, it is possible that, even if you touch the apparatus it was not so cold inside. First of all you have to put your finger in the solution after you stop the transfer to feel if the temperature is cold. With 6% acrylamide it happen that high temperature can be the cause of expansion and gel crambling.

I don't know the molecular weight of you protein but you can try to modify the amp or the time to better optmized the experiment. I use constant voltage so I don't want to give you wrong advice.

A litthe advise. The crambling issue remain with 6% gel, from my experience I always have my gloves wet , and when I have to take the gel (after the running), from the glass plate, I pour the cold transfer buffer on the gel, I wet two 3MM paper sheet with cold tranfer, and then put the 3MM on the gel and I knock over as an omelet this half sandwich. I repeat the same thing after the transfer, I never touch the gel with the gloves, even 10% acrylamide gel, if you have only a small part of the gel or the gloves not properly wet you can break the gel, even the 10%.

The solution was very cold when I was disassembling the apparatus. Thanks for your advice. I wasn't sure if this was a mistake I had made or a common issue.

I've been looking up a lot of troubleshooting techniques for phos-tag gels. One solution I found was to use acrylamide : Bis-acrylamide solution with a higher ratio, such as 37.5:1. We always us 29:1 in our lab. Correct me if I'm wrong, but I understand the higher ratio supposedly produces a more porous gel while maintaining rigidity of a higher percentage gel.

Hi, I found this site that explain really well your question

https://www.nationaldiagnostics.com/electrophoresis/article/polyacrylamide-matrix

Acrylamide forms polymers and Bisacrylamide is the crosslinker of these polymer.

In my lab I always buy the acrylamide : bis-acrylamide solution with a higher ratio 37.5:1 because we performed essentially western blot, I know that the ratio 29:1 is used for analizing both protein and acid nucleic. For your experiment you can search online other protocol to see if suggest different T and C% for acrylamide : bis-acrylamide.

I also find this

https://www.igz.ch/downloads/16079/phos-tagtm_sds-page_guidebook_11.pdf

Troubleshooting from pag 13 to 17

I haven't experience in Phos-tag gels so I am sorry I am not of any help, I only talk about the the cramble and expanding problem and honestly I would try to decrease the amp. I use constant voltage because the constant amp generate more heat and even if you have cold buffer the low % gels are really challenging. ANother observation, the gel is fragile but remain soft or is rigid?

Good luck

Thanks for the links! The first one was very useful. Understanding the ratios will be very valuable in future protocols. I might try switching to 37.5:1 myself based on the chart at the end of the link. Especially for phos-tag gels, because everything migrates slower in those. This might solve my problem.

It's funny how everyone is divided between constant amps or volts. I have been switching between both to try and figure out which I prefer and I have to say I don't notice a difference.

To answer your question, the gel is was very soft and fragile after the transfer.

I have been combing over that second link a lot these past few days, in addition to a few other resources, trying to figure out a way to optimize the phos-tag system for my protein. There are just so many parameters I can adjust and I would like to know what I'm getting into before a try it.

Hi, glad that one of the link is useful for your work, I suppose you have to indentify a high molecular protein and it is not easy. In my experience I also make a gel with glycerol for detect high and low molecular gel in a 4%-12% gel it is not easy at the beginning.

The "costant" debate between voltage and current it would never be resolved, I also try both because I find that probably are both true depending on the experiment (I attached one of the discussion that I found most interesting: https://www.researchgate.net/post/SDS_PAGE_should_be_run_at_constant_current_or_constant_voltage).

If you want to change parameter it is important, as you said, to understand what and why and not changing more than one (maybe two) or you can't figure what's going on.

So summarise the situation for better understand:

1)You run first a 8% gel, you don't say if you transfer it with problem or not...

2) You switch to 6% for improve running time, I suppose at the same condition of 8% and transfer at 380 mA for 5h, is it the ame condition of 8% ?

You change only the gel% is it correct? So I can say that 6% create some problem.

I didn't ask you about the transfer, I mean, without the expansion problem, did you perform a ponceau so you can say that the trasfer is good but you, unfortunately, lost the outermost lanes ?

So I think that as the second link suggest you can improve the softeness of your gel so, it is one of the thing you can do. You can't be constantly afraid to break your gel! I had problem with a 6% for analysis of dystrophin (400KDa) and before using a handmade gel we use a precast and the gel melted and stuck on the membrane. After using a handmade we have to improve the transfer, ON or for 6h at low current.

I would try and underline try to improve the gel softeness and reduce the current or the time of transfer (I will first begin with the current and the time depend on the protein weight). I only add the passage of equilibrate for few minute the gel, at the end of the running, in the cold transfer buffer while you prepare the sandwich.

If you want to run low %T gels, you can stabilise them with 0.5% agarose (electrophoresis grade, that is, low electroendosmosis). This has no effect on separation (pore size of agarose is sufficient for whole virus), but makes the gel less squishy.

Electrophoresis should be performed at constant current (for a minigel, 10 mA for stacking, 20 mA for separation). The reason is that at the beginning of electrophoresis the conductivity of the gel is still high, if you applied a high voltage at this stage, current and hence wattage would be high and the gel would overheat. Limiting the current is effective because as the run progresses and the conductivity of the gel drops, the voltage will automatically increase. At the end of the run, voltage may even become limiting as the power supply may not be able to produce enough voltage to drive the selected current.

For blotting, I had good results with Dunn's buffer (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3) rather than Towbin's, and tank blotters rather than semi-dry.