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Excess acrylamide in wells


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#16 Aris

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Posted 14 May 2009 - 10:30 AM

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



Here comes the recipe:

1.5 ml dH2O
625 ul 0.5 Tris
25 ul 10% SDS
400 ul 30% Acrylamide
12.5 ul 10% APS
5 ul TEMED

#17 Tfal

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Posted 08 July 2009 - 07:39 AM

I used to always make the same % of bis-acrylamide stacking gel whatever was the resolving (sperating) gel's % with (some) successful results.
But then I read in Bio-Rad Mini-Protean's manual a a stacking gel recipe with variable % and figured that it should match the resolving gel's %.
I asked around: some confirmed this assumption while others said that an intermediate % (e.g. 10%) was fine with any resolving gel.

Anyway, now i follow Bio-Rad's instructions just to be safe...

#18 Tfal

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Posted 08 July 2009 - 07:43 AM

and even if it turns out that washing the wells was not your (main) problem, I assume it's always best to do so with ddH2O.
My thinking is you don't want the extra liquid (whether it be acrylamide or not) after polymerizing to mix with samples.

#19 mdfenko

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Posted 08 July 2009 - 08:43 AM

and even if it turns out that washing the wells was not your (main) problem, I assume it's always best to do so with ddH2O.
My thinking is you don't want the extra liquid (whether it be acrylamide or not) after polymerizing to mix with samples.

it's best to use electrode buffer to wash the wells.

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#20 Tfal

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Posted 17 July 2009 - 04:16 AM

Hey mdfenko, thanks for the advice. I guess pH wise it better to use electrode buffer...
Is it good enough if I just rinse the wells briefly with ddH2O and then fill them with electrode buffer when I set my cassette (gels) in the electrophoresis container?

#21 mdfenko

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Posted 17 July 2009 - 07:48 AM

you can do that but water can diffuse into and mix with and dilute the stacking gel buffer.

with electrode buffer you are putting the gel into the same condition in which you will run it.

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#22 gbbg

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Posted 22 July 2009 - 08:27 AM

Hi
I have had the same issue with Biorad Mini Protean while using a 1.0mm spacer and combs combination.

However, I observed that the problem was not so great when I removed the combs soon after the stacking gel polymerized (like within 10min).

We did call Biorad who said they have heard the same prob before, and sent us replacement plates and combs, but the problem persisted when I left the gels+comb for a longer duration (>20-30min)

Hope this helps.


Cheers

#23 Feelcontraire

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Posted 18 November 2009 - 09:25 AM

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


Well, APS-TEMED polymerization takes place in the absence of oxygen.

First: Try adding an amount of gel between the plates so that no extra-gel flows out of the gel when you insert the combs. As this outside gel will be exposed to air and take longer or wont polymerize. Also try increasing APS to 100ul 10% and TEMED 10ul per 10ml of gel. APS activity will fall to half of its activity after a litle time in the fridge or frozen.

Second and highly recommended for everyone: add riboflavin-5-phospate to the stacking gel. Rifoflavin will polymerize in the presence of air, so comb or plates alteration wont matter much, so that you'll have nice formed wells.

Edited by Feelcontraire, 18 November 2009 - 09:30 AM.


#24 mdfenko

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Posted 18 November 2009 - 01:17 PM

First: Try adding an amount of gel between the plates so that no extra-gel flows out of the gel when you insert the combs. As this outside gel will be exposed to air and take longer or wont polymerize. Also try increasing APS to 100ul 10% and TEMED 10ul per 10ml of gel. APS activity will fall to half of its activity after a litle time in the fridge or frozen.

Second and highly recommended for everyone: add riboflavin-5-phospate to the stacking gel. Rifoflavin will polymerize in the presence of air, so comb or plates alteration wont matter much, so that you'll have nice formed wells.

adding more aps-temed will create a gel with shorter strands and will be more fragile.

polymerization with riboflavin (which requires light activation) will be effected by oxygen. it is still a free radical reaction. we use it to polymerize the stacking gel of our non-denaturing page.

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#25 Feelcontraire

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Posted 18 November 2009 - 04:27 PM

First: Try adding an amount of gel between the plates so that no extra-gel flows out of the gel when you insert the combs. As this outside gel will be exposed to air and take longer or wont polymerize. Also try increasing APS to 100ul 10% and TEMED 10ul per 10ml of gel. APS activity will fall to half of its activity after a litle time in the fridge or frozen.

Second and highly recommended for everyone: add riboflavin-5-phospate to the stacking gel. Rifoflavin will polymerize in the presence of air, so comb or plates alteration wont matter much, so that you'll have nice formed wells.

adding more aps-temed will create a gel with shorter strands and will be more fragile.

polymerization with riboflavin (which requires light activation) will be effected by oxygen. it is still a free radical reaction. we use it to polymerize the stacking gel of our non-denaturing page.


Yes, adding more TEMED will make the gel more fragile but as long as it is kept in the 11-1ul/10ml it's ok. Notice it was an advice for people having trouble with there well formation. TEMED also oxidises by time and people sometimes use lower ph gels which reduces TEMED activity for this reason when polymerization doesn't work properly I recommend pointing higher on APS-TEMED values.
If gel fragility is a problem 10% glicerine can be added which gives nice flexibility.

In fact litle oxygen is enough for riboflavin polymerization, but strong degassing may inhibit the polymerization.
You can also use it in denaturing gels, and I strongly recommend it for people that need nicer wells. Also riboflavin is a mild oxidizing agent so it wont add much extra-free radical formation that may interact with proteins in the gel. Riboflavin is added to the standard plus the standard APS-TEMED.

#26 basudec19

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Posted 24 January 2010 - 12:56 PM

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




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#27 JEE

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Posted 08 July 2010 - 08:11 PM

Hi Aris, What u used? Can lend to me as well? Because what i wonder for me is the difficulty to wash the acrylamide in the well.
Question for gbbg, hi, I m also using Biorad Mini Protean (1.0 mm), I having the exactly same problem as u, i also found that it is easy for me to wash if i removed the combs quickly after stacking gel polymerized. i using 10 well comb, theoretically, 44 ul volume of sample can be loaded, but i only able to load 20-25 ul. Should i ask the biorad ppl change for me also? but u say the problem still persisted aft u changed.

#28 asli

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Posted 20 September 2010 - 03:27 AM

Hello. I had the same problem, actually am still having it in every gel. I am sure I use the correct size comb.
The solution I could find is, taking a clean gel loading pipette tip (the ones with extremely thin and long tips), disrupt the gel in the well and try to remove it by kind of stirring inside the well creating a vortex with the pipette tip again.
Works fine with me now, although might take 20-30 minutes to complete, so I suggest you be patient.
Good luck to everyone.

#29 tiki

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Posted 29 October 2010 - 07:58 AM

in fact my problem is not excess acrylamide in well but is refered to wells....
I'm having exactly the same problem as u JEE (Biorad Mini Protean (1.0 mm). i'm using 10 well comb, in theory 44 ul volume of sample can be loaded, but i'm only able to load 20-25 ul. If i need to load more than 25 ul, can i run a few min and reload the sample ?

Edited by tiki, 29 October 2010 - 09:06 AM.


#30 mdfenko

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Posted 04 November 2010 - 08:03 AM

in fact my problem is not excess acrylamide in well but is refered to wells....
I'm having exactly the same problem as u JEE (Biorad Mini Protean (1.0 mm). i'm using 10 well comb, in theory 44 ul volume of sample can be loaded, but i'm only able to load 20-25 ul. If i need to load more than 25 ul, can i run a few min and reload the sample ?

you may be mixing your sample with the electrode buffer when you load the gel. does your loading buffer have sufficient glycerol or sucrose (or something else) to ensure that the sample is significantly more dense than the buffer? are you loading too fast?

talent does what it can
genius does what it must
i used to do what i got paid to do





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