Problem pouring SDS-PAGE gel - (Apr/14/2006 )

I am making a 12 % SDS gel and have a problem. on the side of glass slide after polymerization of gel some buble like blank spaces appear. some time on both side and some time on that side from where i pour my gel.

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hi
clean them with water and dry with ethanol. After setting up the plates and spacer in the pouring apparatus, fill it with ddH2O (also a check of any possibility of gel-escape) and remove it before pouring.
this will slightely hydrate the glass and avoid the bullbes on it.

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When I used biorad system, I never had bubbles, because I was cleaning the plates carefully with ethanol. They were absolutely clean.

now I use novex from invitrogen (because I'm in an other lab..) and it's no more glasses, but single use plastic, and I have for time to time some bubbles, not inside the gel, but between the gel and the plastic : there is no problem of migration. (eventually, the bubbles migrates with the dye front !).

If you have a bubble inside the gel, then it can be a problem. Wash carefully your glass plates (there must be no dust or fibers), and try to pour your gel slower.

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After pouring your gel pour a slight layer of butanol over it. When your gel is solidified, pour off the butanol and pour in the gel for your spacer and put in your spacer. The butanol helps get rid of bubbles on top.

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hi,
1-cleaning of apparatus is very important
2-pour resolving gel slowly in a single stretch with out break (using 10ml pippetman )
3-i wud prefer water to pour on as fred33 says to remove bubbles (pour very gently)

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what do you mean by prefer water to pour on as fred said?
If I'm right, Fred suggested to put water BEFORE to pour with your acrylamide solution. But you should add something else than water ON your acrylamide solution because water is miscible and you would slightly dilute the acrylamide solution on the surface. It is better to use something like isopropanol or butanol. (wahs before pouring the stacking gel).

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Hi missele,
i misunderstood what Fred33 said. i aporgize for that reason. what i want to say in the third point of my previous post is...
after pouring resolving gel i put water (in my ex lab i always used to put water saturated butanol) for proper polymerization.
i was exactly in same impression like u, that if i put water i will dilute the resolving gel slightly. till now i have done severl western blots. i never used butanol always used only water (put water gently n fill the space)n it works absolutely normal. interesting thing is after polymerization u can have straight surface without any bubbles n gaps.
i m completely satisified with this method and quite comfortable with it. i am sorry if i make any diversion in the general impression.

i convinced myself, saying that my resolving gel is 10% and my stacking gel 5%, when i use water may be because of slight dilution it forms a gradient and may allow proteins for proper separation.(my personal opinion)

i just want to share my experience, i donot want to introduce any surprising issues. i wud appreciate your comments.

thanks




[quote name='Missele' date='Apr 17 2006, 05:09 AM' post='48285']
[quote name='payeli' post='48275' date='Apr 17 2006, 01:10 PM']

what do you mean by prefer water to pour on as fred said?
If I'm right, Fred suggested to put water BEFORE to pour with your acrylamide solution. But you should add something else than water ON your acrylamide solution because water is miscible and you would slightly dilute the acrylamide solution on the surface. It is better to use something like isopropanol or butanol. (wahs before pouring the stacking gel).
[/quote]

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Hi Payeli.
I also used water over my acrylamide solution.It was not bad. I was very careful. I never analysed some proteins to close to the surface, then I cannot tell you if it was better...however I'm not convinced. This my opinion.
all the best
Missele

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i mostly use water now (but have used methanol in the past)
- i figure that you if are running your gel for long enough, then your protein of interest should be well away from the stacker/resolver border so it shouldn't really matter. so you get a bit of a gradient - it should be consistant across all the wells anyway....

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hi all ! i doubt the usefulness of methanol as a overlay solution on your resolving gel. methanol is a strong dehydrant and it will concentrate acrilamide solution on the top.
previously water was the option but later switched on to butanol. i personally feel water saturated butanol is a good option. gives a very clean surface without any problem of dilution or concentration of acrylamide solution at the front.
cheers

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