5 replies to this topic

[masi]

I’ve tried running tris-tricine SDS-PAGE gels using 16.5% separating gel (49.5% acrylamide) for peptides below 3kD several times. But i can not polymerize the acrylamide. I have prepared fresh solutions again but still have the same problem. I appreciate any suggestion.


My Recipe: (16.5%)

Acrylamide-bisacrylamide (49.5%T, 3%C):         5 ml
Gel buffer                                                         5 ml
Glycerol                                                            1.6 ml
Water                                                                3.4 ml
TEMED                                                               5 µl
APS (10%)                                                         50µl

[Vini]

masi, on Dec 7 2009, 02:08 PM, said:

I’ve tried running tris-tricine SDS-PAGE gels using 16.5% separating gel (49.5% acrylamide) for peptides below 3kD several times. But i can not polymerize the acrylamide. I have prepared fresh solutions again but still have the same problem. I appreciate any suggestion.


My Recipe: (16.5%)

Acrylamide-bisacrylamide (49.5%T, 3%C):         5 ml
Gel buffer                                                         5 ml
Glycerol                                                            1.6 ml
Water                                                                3.4 ml
TEMED                                                               5 µl
APS (10%)                                                         50µl

are u sure that the APS and TEMED that u r using, are working fine?

[mdfenko]

and, did you degas your solution prior to addition of temed and aps? dissolved oxygen will inhibit the polymerization.

(although, i think your problem may be due to bad aps).

how long have you waited for polymerization? does it eventually polymerize (even a little)?

[masi]

mdfenko, on Dec 8 2009, 12:24 AM, said:

and, did you degas your solution prior to addition of temed and aps? dissolved oxygen will inhibit the polymerization.

(although, i think your problem may be due to bad aps).

how long have you waited for polymerization? does it eventually polymerize (even a little)?

yes i have degas my solution before adding TEMED and APS. it does not polymerize at all. ok i will use a new APS. hope it works.
thanks

[rhythmgenes]

The number one reason for polyacrylamide gel not polymerizing has to be bad APS. i had first hand experience. The lab I was working in could not afford new APS and my prof kept telling me that it made that crackling noise in solution but nontheless APS had gone bad. TEMED though is the catalyst is very unlikely to go bad. Degasing might help; we had luck with with that in the days of depression but not consistent at all.

[anju]

masi, on Dec 7 2009, 12:38 AM, said:

I’ve tried running tris-tricine SDS-PAGE gels using 16.5% separating gel (49.5% acrylamide) for peptides below 3kD several times. But i can not polymerize the acrylamide. I have prepared fresh solutions again but still have the same problem. I appreciate any suggestion.


My Recipe: (16.5%)

Acrylamide-bisacrylamide (49.5%T, 3%C):         5 ml
Gel buffer                                                         5 ml
Glycerol                                                            1.6 ml
Water                                                                3.4 ml
TEMED                                                               5 µl
APS (10%)                                                         50µl

Hi,
I was all the time running Tris-tricine gel for protein separation. What is your gel buffer? I was using Acrylamide from Sigma and for higher concentrations, I used 40% Acrylamide, instead of 30% for my routine gels. For 15% gels (10 ml), I was using 3.3 ml Buffer (3.0 M Tris-HCl, pH 8.45), 100 ul 10%SDS, 4 ml 40% Acrylamide/Bis-acrylamide (Sigma), 2 ml 50% Glycerol, 50 ul 10% APS, 5 ul TEMED and 545 ul water.
You can try this recipe. I modified this recipe from the original and always got sharp bands.

Anju

Edited by anju, 14 December 2009 - 12:24 PM.