Excess acrylamide in wells - (Apr/20/2009 )
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
Stupid question!
Did you try wash the wells before loading your samples?
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.
BadKarma on Apr 21 2009, 04:49 PM said:
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
bob1 on Apr 22 2009, 08:16 AM said:
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
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).
mdfenko on Apr 22 2009, 11:23 PM said:
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
??????? Isnt it Badkarma that should apologize ??????????????
Rather nasty to start your response saying that it is a stupid question
BadKarma wonīt appologize.
I forgot wash the wells lat week and i didnīt understand what happen to my gel.
Ok...
I'm sorry if I offended you
Lusen on Apr 23 2009, 04:18 AM said:
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...".