If your acrylamide/bis solution had been stored at 4°C, there must be no problem: it is quite stable over time. Check out your persulfate solution: this one, on the contrary, is very unstable, and usually it is old persulfate which is the main reason for poor polymerisation. Just try a freshly prepared solution. Check out your TEMED as well, although, if stored at 4°C, it should not be a problem.

Polymerisation can be boosted by exposing the gel to moderate heat and light (due to the radical mechanism of the reaction). I routinely used a regular table lamp with a yellow light bulb (like the ones we often have at home) positioned within several cm from the gel to accelerate PAAG polymerisation (full solidification within 5 min).

If your acrylamide/bis solution had been stored at 4°C, there must be no problem: it is quite stable over time. Check out your persulfate solution: this one, on the contrary, is very unstable, and usually it is old persulfate which is the main reason for poor polymerisation. Just try a freshly prepared solution. Check out your TEMED as well, although, if stored at 4°C, it should not be a problem.

Polymerisation can be boosted by exposing the gel to moderate heat and light (due to the radical mechanism of the reaction). I routinely used a regular table lamp with a yellow light bulb (like the ones we often have at home) positioned within several cm from the gel to accelerate PAAG polymerisation (full solidification within 5 min).

Mostly the problem could be poor ammonium persulfate & TEMED quality. As a trial you could try adding all the ingredients of the resolving gel except the persulfate solution & then try adding a few crystals of the persulfate salt directly to the solution & leaving it. If it polymerizes then maybe the APS is OK, only the solution needs to be freshly prepared for your gel.

Hi there,

As mentioned above, polymerization rate seems very low so repeat the gel preparation with new batches of TEMED or/and Ammonium Persulfate (the latter being more prone to degradation when in solution).

Thank you, PhD. Smirnov, PhD. Menon and PhD. Liger.

My acrylamide is powder. when I store at 4oC, they were balled up and probably they absordbed water.

I checked my Persulfate and TEMED, they are ok. They were stored at 4oC. They will be newly prepared in every time I make a gel. Gel still polymerized. Only when I separate gel from the plate and rinse it by water or put it into staining solution, the gel is degraded and dissolved.

Especially, during electrophoresis, there is no band appeared, even Protein Ladder. or they were spreaded in to two sides, instead of one line.

I think Problem is acrylamide.

Can I make a acrylamide gel without bis-acrylamide?

Without bis it does not make sense: you will only get linear polymerisation without branching, meaning that there will be no real pores in your gel. Are you sure about the proportion between acrylamide and bis? Should be 37,5:1 for SDS-PAGE.

Check the pH of the solutions used

check ur acrylamide solution first prepare fresh solution and store it on 4 degree. and also temed may not work ,use aps freshly prepared it may be the possible reason behind ur problem.

thank u regards

Hi,

You say your gel polymerizes but when you separate it from the plate it dissolves..How can you be sure your gel is polymerizing well? Sometimes, the upper surface might look polymerized, but the gel as a whole is not polymerized well. That could be why your protein ladder did not run either.

I agree with most comments above, if your acrylamide solution has the correct ratio of bis:acryl and is stored at 4 degrees, it should be ok. APS is more likely the cause of improper polymerization (APS solutions are susceptible to degradation if stored improperly). As Alexander has pointed out, environmental conditions have a role to play in polymerization - make sure its neither too cold not too hot when you are polymerizing the gel, moderate amount of heat is fine. Also, check the pH of your Tris buffers pH 6.8 and pH 8.8 - make sure you are not using pH 6.8 buffer for the resolving gel :) One undergrad trainee in our lab did that once..honest mistake - happens with all of us!

Good luck! I hope you can resolve your gel issues soon :) (pun intended)

Dear PhD Smirnov

Thank you for your sharing.

I only use acrylamide, not bis – acrylamide. Is it Wrong, sir?

Dear PhD Cardozo

Thank you for your advise. I will check them again.

Dear Ms. Rani

Thank you. I will freshly prepare my APS solution.

Dear Ms. Bajaj

Thank for your advises. I really like your the last sentence :) it’s so cool. You must have a good sense of humor :). I also hope what you hoped.

Maybe, many mistakes have been happening to me :)))

Dear Ms. Bajaj and PhD Smirnov

Acrylamide and APS  are powder formation. When I use, I resolve it into water. I store at 4oC.

Polymerization happens at room temperature.

I only use acrylamide, not bis – acrylamide. Is that problem?

Normally, people only need about 5% for Stacking Gel and 12% for Resolving Gel, but I need 35%. Do you feel this abnormal?

Thank you all so much

welcome 

all the best 

regards

You must use bis-acrylamide, without it your gel won't polymerize. Acrylamide itself will only make linear polymers, you need bis-acrylamide as a cross-linker to form a gel. Here's a link that can help you calculate the amount of bis-acrylamide you'll need to add:

http://www.bio-rad.com/LifeScience/pdf/Bulletin_9413.pdf

Dear Ms. Bajaj

Oh I see. Thank you so much for your advice. I will do it.

Best Regards

APS should be fresh 

An additional comment: Acrylamide is not very healthy, especially not the powder form due to the dust. For your own safety either buy pre-diluted acrylamide solutions (they are in most cases already a mixture of AA and Bis-AA) or work in a hood when weighing the powder. The least thing you can do if you don't have the equipment is to use these one-way masks that doctors use.

Many of the reagents employed in the 2D and 1D electoforesis have risks to human health. Be careful and always wear gloves

Hello Phúc,

Here is the acrylamide:bisacrylamide solution we utilize in our lab: https://www.fishersci.ca/productDetails_23908

I have also attached my recipes for making gels. Note that this is for a 30mL gel, which you can obviously scale down to meet your needs. Also, we traditionally freeze aliquots of 10% APS and dispose of after use. Never had a problem but I would not recommend refreezing and reusing the leftover.

I hope you would have find your solution by now, but if you don't i would suggest you following things;

1) Add appropriate amount of Acrylamide /bis-Acrylamide and store the solution at 4C.

2) Prepare fresh APS and store that too into the fridge if you want to use in next couple of days.

3) Keep the gel assembly in 37C for 10-20 min, that enhances the polymerisation.

Hope this will help.

Good luck.

Dear Ms. Asthana

Thank you so much.

Dear Mr. Grotelueschen

Thank you for your advice.

Dear PhD. Cardozo

Thank you for your recommendation.

Dear Ms. Dzialo

Thank you so much. I will use your recipes when  I get new acrylamide:bisacrylamide solution.

 Dear PhD. Pandey

Thank you for your help. I will do it.