I 've used 0.1 ml of 10% aps to prepare 10 ml separating gel, but it was not solidified again tried with .03 ml of 25% aps, but not solidified. APS was freshly made but not solidified after waiting one and half hour. What could be the problem and what percentage and what amount can I use for separating and stacking gel, to see protein expression.
Make sure the APS is not out of date and has no hole in the tub. I found this out when I first started western blotting.
Could you post the recipe that you used for your gel? I would like to see how much and what kind of acrylamide you used.
Make sure it is APS for electrophoresis, in the case of Sigma APS it says on the bottle. Once I've mistaken the bottles and used the one that did not say it was for electrophoresis and it did not polymerise.
Please mention the TEMED concentration and check the date of purchase and expiry date.. What's the percentage of gel and SDS.. All the best friend..
Here is the detail of concentration what he used: for 10 ml (Resolving gel)
1. H2O : 3.3 ml
2. 30% Acrylamide mix : 4.0 ml
3. 1.5 M Tris (Ph 8.8) : 2.5 ml
4. 10%SDS : 0.1 ml
5. 10% APS : 0.1 ml
6. TEMED : 0.004 ml
Thank you all
Increase the volume of TEMED to 0.008ml and check. Also make sure that APS and TEMED is not out of date
I assume that the volume of the added TEMED is far too low. We use 50 µl TEMED and 100 µl 10% APS (for a 15 ml gel) and the gel polymerises within 10 min.
Baskaran and Anja are right. Just increase your TEMED volume and your gel will polimerize easily. I use 33uL TEMED and 100uL 10% APS. Gels are ready in 10-15 min. Good luck!
Another thing to check is the proper storage of APS: APS is hygroscopic and is ideally stored in a dessicator. If you see that the powder seems to have absorbed some moisture, you should buy a new stock and try with that. I also agree that the amount of TEMED is low. I used 20 µl in 10 ml total volume of gel and it worked perfectly.
I use the same recipe as yours but I use 10-12 µl of TEMED. And yes the APS should be in good condition. It is hygroscopic and readily degrades. The container should be air-tight. I had faced the same problem, changing the APS solved the problem.
A rather "silly" suggestion,but actually self experienced...Once we found out that the person making the APS had mistakenly taken ammonium sulphate and not ammonium persulfate...took some time to figure that out...
I use temed and APS in ratio 1 of temed for 15 of APS 10% ( 6ul of temed 100% and 90 ul of APS 10% for a gel. I prepare the gel mix in ice and after ten minutes at RT the gel is polymerized!
As APS is so cheap, you should probably buy some new. We always store our APS in measured alloquotes for 1mL. Just measure the powder into a 1.7 mL tube and store in a minus 20 until you need it. Then add 1mL of water. This stretches out the APS and makes sure it is fresh.
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