SDS-PAGE over heating and will western work from that gel? - sds page running and western blot of gel run under too much heating (Aug/16/2006 )

hi all


i have a silly doubt ,, i dont have much time to repeat the experiment ,,, hence this long story and silly qn

i ran sds page gel,, the gel casting was good,, no bubbles and it set nicely even the arms of the well were very good in shape ,,,
then while running i noticed that there is some sort of bubbles or tunnels like in that you see the ants digs viewed through side ways ,,, hope you got some picture ,,,,


they form near the bottom of the gel

and results in slow running of the gel plus heating up


i run the gel at constant current 20 mA for one mini gel adn 40 for two
usually the voltage is around 100 or soemthing

but this case it rose to 400 V ,, thats how i found that there was resistance and the dye band was yellow in colour !!!!!!!

after destaining the gel i saw the distorted bands

can i use this gel for western ? will the overheating affect the protein ?
will antibody work ??

anyone please

regards
laxmi

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Is it worth to try?
what happened to me once was a boiling transfer.
the transfer was fine, detection of proteins was fine, but not phospho-proteins.

from time to time I also see bubbles, between the gel and the glass plate.
However it has no incidence on the migration or the voltage.

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May be someting wrong with the running buffer...make fresh buffer.

The bubble is due to the overheating of the buffer (the gel expand in heat) and nothing wrong with the gel.

I hope this may help.

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hi

thank you minnie mouse and missele

i noticed that it could be that while inserting the comb after removing the ethanol on the resovling gel that the bubbles get introduced

and it could be the buffer too , i prepare fresh from the stock !! still someone else made the stock so have to check that

thanks for the suggestions

i will try to run the gel in 4 degree and see

regards

laxmi

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hi laxmi

proteins can be transferred even if the gel has heated up a little, since you are not concerned with enzymic acyivity in situ, and can be processed for elisa.

what i understand is that you don't have bubbles but ramifying thread like things. my guess would be that the gel at the base is no longer firmly stuck to the glass plate and a space has developed. this can happen from unclean plates in the first place. i do not know if you are using one glass and one alumina plate, or both glass plates.

its a silly suggestion,but could it be that by mistake the running buffer was used undiluted?

- viv

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hi viv

i use one alumina and another glass
i washed with soap and water and then rinsed with distilled water
after that wiped with kimwipe

but the wiping could have left some dust behind from that tissue mmm could be that

i take 75ml of 4X tris glycine add 3 ml of 10% sds and make upto 300ml for two mini gel Hoefer i think ,,
other people use the same buffer no problem i think ,,

i tried sds today again, only one gel ( the western no band !!)
no prob went nicely till the end ,, at the end some increase in voltage and the system shut down
couple of times stil success

i guessed one other option dont konw if its valid or not
last time i ran two gel at 40mA , one on each side of the stand
but my desk is directly in the path of the air conditioner draft ,,,, so i was thinking maybe one side was cold and the other one was not

could it have any effect ?

thanks for the reply

regards
laxmi

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No risk, it's very difficult to load your sample if the wells are already loaded with a 10x running buffer.

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I don't understand why it's going up to 400V.
for security purpose, you should set the electrophoresis apparatus to 200V so that it can't increase so much, and I would prepare again the running buffer.
20 mA per gel is fine (40 mA if you have two gels)

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ok laxmi

1. you have to use one alumina and one glass or else you wont see the wells nor the migration. always wipe the plates, both glass and alumina, with ethanol using kimwipes immediately prior to gel casting.

2. i also use the hoefer mini gel system and i love it. nothing goes wrong anywhere. all hoefer assemblies come with a built in channel in the middle of the unit for cold water circulation to cool the upper buffer and the gel through the alumina plates and minimize heat. use a water circulator. in case there is no circulator (like the multitemp II of lkb/pharmacia/amersham) you could try to connect the assembly with the tap running water. however, if everything else is ok, the gels frankly don't get heated up so much even without water circulation.

3. i would suggest that you use 0.75 mm gels, and not thicker. did you run the gel at 4c, what happened?

4. run two gels at about 150v constant voltage mode. the current will be adjusted suitably depending on the resistance offered by the gel.

5. i am wondering if there is something wrong with the power supply itself. try another supply, and ask the workshop to check out this one for correct and stable output.

good luck

-viv

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well maybe you shoul have used more buffer in upper tank, or replaced whole tanks at half run...
i think western will work (but for phospho proteins, i think phosphate is out... )

not directly related, but my boss did an experiment with RNA (and i'm not too sure he did with proteins so i just mention for RNA...) in an acrlamide gel he reguary increased the voltage and he said to me it was relative hard to touch the glass as it was hot...
and the experiments were fine !

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