Problem with "smiling effect" on SDS-PAGE gel - (Mar/27/2009 )
Hi everyone, i ran couple SDS PAGE in these few weeks.. and i am using Tricine SDS PAGE system, because one of my target was 4k.
I am using 16% gel, and the formula is the following
AB 50% 5ml
Gel Buffer 5ml
Glycerol 1.5g
Add water to final volume 15ml
APS 10% 50 microl
TEMED 5microl
Anode buffet = 1M tris, 0.225M HCL, pH 8.9
Cathode buffet = 1M tris, 1M tricine, 1% SDS, pH 8.25
Gel buffer = 3M tris, 1M HCL, 0.3 SDS, pH 8.45
All buffers are freshly made yesterday
I ran at initial 30V, after the protein enter the stacking gel, i increase the voltage to 190V (i use constant V)
i have totally 10 lanes, and the smiling effect was always occurred at around lane 1-2 and lane 9-10, and the lanes in between are ok. I also put the whole system in ice while i preforming the run.
I have two questions..
1) how to get rid of the smiling effect ?
2) Will the glycerol cause the smiling effect?
For question 2, i asked because i did try to use the same buffer, but i didnt add the glycerol, therefore, i am wondering the smiling effect was caused by the glycerol...
Can anyone help me ... thanks a lot .....
do not let empty wells beside your sample, load them with the same buffer than your sample
little mouse on Mar 27 2009, 07:01 PM said:
I did load all the wells with my samples....
Danny Chow on Mar 27 2009, 08:00 AM said:
little mouse on Mar 27 2009, 07:01 PM said:
I did load all the wells with my samples....
try running at lower voltages, 100 Volts until they reach the separating gel and then bump it up to max. 150 but no further. Tell me if you had any luck
Aris on Mar 27 2009, 03:27 PM said:
Danny Chow on Mar 27 2009, 08:00 AM said:
little mouse on Mar 27 2009, 07:01 PM said:
I did load all the wells with my samples....
try running at lower voltages, 100 Volts until they reach the separating gel and then bump it up to max. 150 but no further. Tell me if you had any luck
This might be a very "unscientific" advice.... but I'd just buy pre-cast gels. They always look good, you never get smileys when you run them.
It's not so horribly expensive (about 5pounds/gel) and you can spend your time solving problems which are actually important.
This might be a very "unscientific" advice.... but I'd just buy pre-cast gels. They always look good, you never get smileys when you run them.
It's not so horribly expensive (about 5pounds/gel) and you can spend your time solving problems which are actually important.