31 replies to this topic

[Rob Steuart]

Having issues with SDS-PAGE. Have recently moved labs and i am now using teh 8cm Protean system, was previuously using teh Larger 16com system.

When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.

Any help would be great

Cheers

Rob

Edited by Rob Steuart, 20 April 2009 - 08:58 PM.

[BadKarma]

Stupid question!

Did you try wash the wells before loading your samples?

[bob1]

Also make sure that you have the right comb for the right spacers (i.e. 0.75mm comb for 0.75mm spacers), the combs look identical at a casual glance for the different thicknesses, and will sit nicely in the sandwich, but wells will not form properly with a thinner comb than spacer.

Actually the wells will form, it is just that there is a flap of acrylamide that falls in and blocks you from loading into the well.

[Rob Steuart]

BadKarma, on Apr 21 2009, 04:49 PM, said:

Stupid question!

Did you try wash the wells before loading your samples?

Gee wash the wells never thought of that

Do you think i would put a question up like this if i hadn't tried evrything i can think of.

I hope that bad karma come back to at some point

[Rob Steuart]

bob1, on Apr 22 2009, 08:16 AM, said:

Also make sure that you have the right comb for the right spacers (i.e. 0.75mm comb for 0.75mm spacers), the combs look identical at a casual glance for the different thicknesses, and will sit nicely in the sandwich, but wells will not form properly with a thinner comb than spacer.

Actually the wells will form, it is just that there is a flap of acrylamide that falls in and blocks you from loading into the well.

I am using old equip and the spacer dont have labels. Have aligned the combs and the spacer and they appear to be the same width. Will jsut percivere

Cheers

[mdfenko]

as our apparatus aged the plates tended to warp (slightly, not very noticeably, if at all). we would get skins in the wells which we would clean out with a spatula.

sometimes we would clamp the plates at the teeth of the comb to avoid the skins (if we could).

another source of interfering gel is at the top of the plate. sometimes some gel will polymerize at the top, between the cut and the comb on the edge. this can slide into the well and interfere with loading. we would clean the top of the cut after removing the comb (before adding buffer) to avoid this problem.

by the way, you didn't have to be nasty in your response to bad karma. sometimes even the most experienced miss the obvious (we have seen it often in these fora).

[Rob Steuart]

mdfenko, on Apr 22 2009, 11:23 PM, said:

as our apparatus aged the plates tended to warp (slightly, not very noticeably, if at all). we would get skins in the wells which we would clean out with a spatula.

sometimes we would clamp the plates at the teeth of the comb to avoid the skins (if we could).

another source of interfering gel is at the top of the plate. sometimes some gel will polymerize at the top, between the cut and the comb on the edge. this can slide into the well and interfere with loading. we would clean the top of the cut after removing the comb (before adding buffer) to avoid this problem.

by the way, you didn't have to be nasty in your response to bad karma. sometimes even the most experienced miss the obvious (we have seen it often in these fora).

Thanks and i apologise for my ealrier post

[Lusen]

???????  Isnt it Badkarma that should apologize   ??????????????

Rather nasty to start your response saying that it is a stupid question

[BadKarma]

BadKarma won´t appologize.
I forgot wash the wells lat week and i didn´t understand what happen to my gel.
Ok...
I'm sorry if I offended you

[mdfenko]

Lusen, on Apr 23 2009, 04:18 AM, said:

???????  Isnt it Badkarma that should apologize   ??????????????

Rather nasty to start your response saying that it is a stupid question

it's my understanding (of course, i could be wrong) that bad karma was calling his (her?) own question stupid.

we often preface questions of the elementary and/or obvious with "this may be a stupid question but..." or "i don't want to insult you but...".

Edited by mdfenko, 23 April 2009 - 07:05 AM.

[BadKarma]

I should had write "this may be a stupid question but..." but I'm a lazy person.  

And my question could be stupid because was the first thing I thought when I read the post and my first thoughts... well you saw what happen when I said them.  

Apologize the misunderstanding.

By the way it's her    lol

[Rob Steuart]

BadKarma, on Apr 23 2009, 11:33 PM, said:

I should had write "this may be a stupid question but..." but I'm a lazy person.  

And my question could be stupid because was the first thing I thought when I read the post and my first thoughts... well you saw what happen when I said them.  

Apologize the misunderstanding.

By the way it's her    lol

No dramas.

The exclamation mark threw me

Cheers

Edited by Rob Steuart, 24 April 2009 - 12:12 AM.

[sudhakar mutyala]

Hi

Try to wash the comb with Methanol before you insert into the plate , Add little extra TEMED and APS. wash the wells with water before you use them.

Hope it will solve and try to use the same spacers and comb of same size.

Regards
Sudhakar

Rob Steuart, on Apr 20 2009, 08:57 PM, said:

Having issues with SDS-PAGE. Have recently moved labs and i am now using teh 8cm Protean system, was previuously using teh Larger 16com system.

When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.

Any help would be great

Cheers

Rob

[Aris]

Rob Steuart, on Apr 20 2009, 09:57 PM, said:

Having issues with SDS-PAGE. Have recently moved labs and i am now using teh 8cm Protean system, was previuously using teh Larger 16com system.

When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.

Any help would be great

Cheers

Rob

Had the same problem as you couple of months ago, tried to wash the wells with dH2O but no good, the advise was all the same, wash the wells, i also placed the comb in Methanol. No use. Till i found a new recipe for the stacking Gel. Can send it to you if you still have problems

Cheers mate

[Rob Steuart]

Aris, on May 8 2009, 07:29 AM, said:

Rob Steuart, on Apr 20 2009, 09:57 PM, said:

Having issues with SDS-PAGE. Have recently moved labs and i am now using teh 8cm Protean system, was previuously using teh Larger 16com system.

When i remove the comb from my stacking gel there is excess acrylamide in the wells blocking me from loading sample. The combs i am using fit snugly into the gel casette so not sure where the problem is arising. I never had any issues in teh larger system, has only started since i began using teh smaller system.

Any help would be great

Cheers

Rob

Had the same problem as you couple of months ago, tried to wash the wells with dH2O but no good, the advise was all the same, wash the wells, i also placed the comb in Methanol. No use. Till i found a new recipe for the stacking Gel. Can send it to you if you still have problems

Cheers mate

Thanks Aris

recipe would be great

cheers