11 replies to this topic

[Danny Chow]

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 .....

[little mouse]

do not let empty wells beside your sample, load them with the same buffer than your sample

[Danny Chow]

little mouse, on Mar 27 2009, 07:01 PM, said:

do not let empty wells beside your sample, load them with the same buffer than your sample

I did load all the wells with my samples....

[Aris]

Danny Chow, on Mar 27 2009, 08:00 AM, said:

little mouse, on Mar 27 2009, 07:01 PM, said:

do not let empty wells beside your sample, load them with the same buffer than your sample

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

[dedee]

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:

do not let empty wells beside your sample, load them with the same buffer than your sample

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.

[Aris]

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.
[/quote]


I agree, but the problem shouldnt be the gel actually...and after all smilies and sad faces when running a gel are NOT that dramatic, it is just that the lanes will be shifted. I instist on the voltage, if this doesnt work we have to think again

[Danny Chow]

Thank you guys !!!
i would try lowering the voltage and manage the temp. better....

reli reli thanks!!!

[Danny Chow]

I tried 150V and run the whole thing in ICE... but still ... smiling effect...

would it be the temperature? Should i not running the whole thing in ice??
cause most of the ... i mean all of the protocols didnt suggest me to run it at low temperature....

except buying a commercial gel, is there any other ways to solve the problem ?

[MaggieRoara]

Danny Chow, on Mar 30 2009, 12:35 AM, said:

I tried 150V and run the whole thing in ICE... but still ... smiling effect...

would it be the temperature? Should i not running the whole thing in ice??
cause most of the ... i mean all of the protocols didnt suggest me to run it at low temperature....

except buying a commercial gel, is there any other ways to solve the problem ?

the smiling effect is most probably due to heating. Could be that you did not mix the gel properly before pouring. There, there is a resistance difference throughout the gel, heating up some place and not others.
Or it could just be that your gel is uneven in thickness, use new gel plates and try it out.


whenever i see even a hint of smiles on my gels, I turn down the voltage till the dye front straightens out and then resume running at normal voltage

[scoob00]

Hi!

I run high percentage Tricne gels all the time, but I never go up to 150V!! Never ice. I run constant 60V through the stack, 80V through the rest. Takes a few hours but it's worth it.

Hope this helps...

[Danny Chow]

MaggieRoara, on Mar 30 2009, 05:24 PM, said:

Danny Chow, on Mar 30 2009, 12:35 AM, said:

I tried 150V and run the whole thing in ICE... but still ... smiling effect...

would it be the temperature? Should i not running the whole thing in ice??
cause most of the ... i mean all of the protocols didnt suggest me to run it at low temperature....

except buying a commercial gel, is there any other ways to solve the problem ?

the smiling effect is most probably due to heating. Could be that you did not mix the gel properly before pouring. There, there is a resistance difference throughout the gel, heating up some place and not others.
Or it could just be that your gel is uneven in thickness, use new gel plates and try it out.


whenever i see even a hint of smiles on my gels, I turn down the voltage till the dye front straightens out and then resume running at normal voltage

O.. thanks
But the smiling effect was not happened on the sample (dye front), it is actually happened on the gel itself..
at the end of the gel, it is kind of changing its shape by forming a bump around the end of the gel, blocking the dye front to go down further....

[lab rat]

O.. thanks
But the smiling effect was not happened on the sample (dye front), it is actually happened on the gel itself..
at the end of the gel, it is kind of changing its shape by forming a bump around the end of the gel, blocking the dye front to go down further....
[/quote]


I have had this problem as well. It's like the gel is melting near the bottom of the gel, and everything stops at the melted part. I use only purchased gels. Running on ice and using a stir bar seems to lessen this effect. Running at a lower voltage does also.

The melting problem seemed more pronounced (for my stuff) when I used two differently-pH'ed running buffers as you are. Could differences between the pH or ionic strength of the gel and buffers be the issue?

lab rat