Problem with 15% SDS page gel during electrophoresis - (Sep/10/2014 )
I am facing a problem while running electrophoresis on a 15% SDS PAGE gel for a Western Blot.
My goal is to study the expression of a protein weighing about 16 kDa, so I chose a 15% resolving gel (and a 5% stacking gel)
Everything goes on well, as I prepare it, load my samples and run the electrophoresis (150V, 1h-1h30).
However, as I reach the end of the gel (and I need to, so I can see the protein I am looking for), the gel starts over polymerazing, creating a kind of pudding, the bromophenol bleu turns yellow, and my sample cannot migrate anymore. So I end up not seeing my protein...
Has someone ever faced this problem? What would you suggest?
If you need more information:
I prepare my resolving gel as follow (final volume: 10mL)
Water: 2,3 mL
30% acryl-bisacrylamide mix: 5mL
1,5M Tris, pH 8.8: 2,5 mL
10% SDS: 0,1 mL
10% Ammonium Persulfate: 0,1mL
My electrophoresis buffer is prepared as follow (10X, then diluted in water)
15 g Tris
72 g Glycine
5 g SDS
in 500 mL water
how long do you allow your gel to polymerize?
you are not experiencing "over polymerization" (i've never come across this). this is often seen when the sds is old and decomposing. try a fresh lot of sds (see if you can get some from a different lab).
15% gels can be tricky. for 16 kDa we would use 10% tris-tricine sds-page.
Thank you for your answer!
My gels polymerize during about 20 minutes. I always have an extra I don't use, I just leave it in my tube an wait for it to polimerize. When it's done, I consider the gel within the glass is polimerized.
I will try another SDS tomorrow then, I hope it's gonna be better!
Are you sure about using a 10% gel? I have been told that it didn't allow to separate proteins smaller ther 20 kDA...?
Have a nice day!
using tricine instead of glycine changes the range of the gel. it favors lower molecular weights.
also, 20 minutes is not long enough for the gel to "cure" properly. there may be some incompletely polymerized acrylamide left in the gel.
I have tried with a new SDS but the result is the same...
Do you have a recepie for your 10% tris-tricine sds-page? I don't get how you do this since there is no glycine in my recepie, but I would be pleased to try it!
Thanks a lot!
this post includes the formulation for tris-tricine gels in one of the posts (as a downloadable rtf document): http://www.protocol-online.org/forums/topic/26104-blurry-bands/?hl=+tricine++#entry137266
what recipe are you using?
Thank you very much
It seems that I don't have the required equipment to run the gel... I have to check this out with my fellow workers!
I will try it as soon as I can.
Thank you again
i just noticed that you posted the recipe in your first post. you have glycine in the electrode buffer. the glycine is replaced with tricine for the tris-tricine gels.
what equipment do you not have? the gels are run in the same apparatus. you just have to make up some new solutions (unless you don't have tricine, maybe you can borrow some?).