My separating gel is not getting polymerized. (It does not set). I am following protocol by Schagger, Von Jagow protocol. Can anyone please forward me your protocol for Tricine SDS PAGE?
Hello!!
i used to work with this protocol. The ammonium persulfate solution should be made in the day (it`s very important!)
This is the protocol:
http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html
Good luck!
hi abheepsa.
you can try this composition as it always worked in my lab while casting gel for tricine.
The composition is:
water 3.35 ml
separating gel buffer 5 ml
separating gel acrylamide (2x) 5 ml
glycerol 1.6 ml
APS (10 %)freshly prepared 150ul
TEMED 10 ul
composition for separating gel buffer
3 M tris base, 0.3% SDS. dissolve and adjust pH to 8.9 using HCl. make up the volume 100 ml.
composition for separating gel acrylamide (2X)
acrylamide 48g
bis acrylamide 3g
make up the volume 100 ml.
I hope this will work.
I have prepared my APS 3 months ago and is still working fine. I don't get any problem in gel polymerization.
Suggestions given by others should solve your problem but in case those do not work you can increase the amount of APS and TEMED. You should also check the TEMED whether it is too old or freshly procured. For an alternative protocol for Tris-Tricine PAGE, you can consult the protocol given in Chapter 10 in Current Protocols in Molecular Biology by Ausubel.
use 10% APS always freshly prepared and add in order. The order is SDS +APS+TEMED. Make sure TEMED should not get too old and smelled properly.
Wash your gel plates properly and Prepare APS freshely, Check contamination your TEMED and Acylamide and Bis acrylamide.
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