I am trying to prepare the SDS PAGE gel. But the problem is the separating gel is highly sticky and does not look like a regular gel. Then apart from this the stacking gel is not getting polymerized. Below mentioned is the composition:
1. Separating gel: (10ml, 12%)
Water: 3.3 ml
30% Acrylamide: 4 ml
Tris Buffer (pH 8.8): 2.5 ml
10% SDS: 100 microliters.
10% APS: 100 microliters
TEMED: 50 microliters.
2. Stacking gel: (3ml, 5%)
Water: 2.1 ml
30% Acrylamide: 0.5 ml
Tris Buffer (pH 6.8): 0.38 ml
10% SDS: 30 microliters.
10% APS: 30 microliters
TEMED: 20 microliters.
Please suggest what could be the problem and how it can be mitigated ?
From my side I have changed the tris buffers even then the problem remains.
Gel are mixture of Acrylamide and bisacrylamide. What is the % of bis in your gels.
The APS must be fairly fresh or it will not react with the TEMED and your gel will not polymerize. Either make it up fresh each time or make a few small aliquots and freeze them and use them in a month or two.
It should be a mix of Acrylamide (30%) and bisacrylamide (0,8%), not only acrylamide. Acrylamide alone forms linear polymers. The bisacrylamide introduces crosslinks between polyacrylamide chains. The 'pore size' is determined by the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of bisacrylamide to acrylamide and a high acrylamide concentration cause low electrophoretic mobility.
Thanks for your response Richa Tyagi, Christa Montgomery and Valeria Marrocco.
I am using 30% Acrylamide (29% Acrylamide and 1% bisacrylamide).
I am using 10% APS fresh always.
I am using Ultrapure Water.
You put too many TEMED. For your separating gel, put 5 uL TEMED and 4 uL TEMED for the stacking gel. For the stacking gel, I used 50 uL 10% SDS and 50 uL 10% APS. Wait 15 - 20 minutes for the acrylamide to polymerized.
Hi Try to cut down volume of Temed which may cause sticky. Both try half the volume.
For the case of separating gel, I think you put too much TEMED that's why it is so sticky. For 10 mL of gel, I usually used 20 uL of TEMED.
For the stacking gel, try to reduce the volume of water and increase the Tris buffer and 10% SDS.
What is the concentration of your Tris buffer?
Prepare both the tris buffer freshly (keep a close eye on the pH). Also for 10 ml Resolving (12%), use only 4-6 microlit of TEMED. And 3 microlit for 3 ml stacking gel (5%). I believe it would solve your problem.
I use this recipe and works fine: (for 2 gels)
Resolving gel:
6.7mL 30% acrilamide
8.1mL dH2O
5mL resolving buffer
200uL 10%SDS
200uL APS
15uL TEMED
Stacking gel:
1mL 30$ acrilamide
2.5mL dH2O
1.25mL stacking buffer
50uL 10%SDS
50uL APS
5uL TEMED
hope it'll help.
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