The SDS PAGE was ran at 80V and after 5 minutes the loading dye and sample(s) are leaking (see attached picture). What can be the problem and suggestions for avoiding it. Thank you very much in advance.
It looks like the stacking gel has come away from the plate perhaps due to drying out - was this a prepacked gel or cast by yourself?
Do you have stacking gel?
It is look like your sample come into separating gel after short time.
You might have incorrect buffers in stacking/separating gels or in the tank!!!
Dear Niraj
Clean well the glass plate and Let the gel polymerize for hours
Regards
Hi Marcus,
I cast my gels, it was over laid with ethanol and ethanol was removed after 30 minutes. Then I poured the stacking gel.
Thank you!
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Hi Alireza,
Yes, I have stacking gel.
Thank you!
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Hi Aamal,
All the buffers are correct because the other people in the lab are using the same reagents. Their gels are coming out nice.
Thank you!
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Hi Nawroz,
Good suggestions. I usually keep gels polymerizing for longer time.
Thank you!
Did you use them immediately or store them? In the past I've stored the gels in the fridge but they've still dehydrated a little where I've not totally sealed the cling film. Perhaps the APS/TEMED didn't mix thoroughly and some of the gel didn't polymerise in areas? I'll bet it's a one off and you won't see it again!!
If the leakage does not occur before running the gel and applying voltage, it seems that this is due to high voltage and current that is used or unpolymerized gel. I also suggest that do not overlay your seoerating gel with ethanol. Instead use water or water-saturated isobutanol.
When I cast my own gels, I use a 200 ul buffer overlay and store them in the cold room overnight. Next day I pour the stacking gel and use anytime after 20 mins but less than 2 hours. It may help if you handle the gel more delicately, care must be taken not to squeeze the gel.
Overlay with water-saturated isobutanol has worked well for me.
If in the gelbox not just so and clamped pressure can bend the glass
As suggested by others make sure the gel isn't dehydrated. I would also suggest making up fresh buffers and thoroughly cleaning out the box. Sometimes if you put too many wells in the gel when making them they are more prone to leaking.
There will be a "tiny hole chamber" formed between the gel surface and its covering glass side when the gel is deformed or dehydrated. The loading dye will penetrate into the hole chamber when it reaches. One possibility is that the gel was deformed by force when removing the ethanol.
Just to follow up, I could not find the answer why is it happening but I am getting proper bands on the blots. So, for now it least likely a problem.
Thank you very much for your replies and it helped a lot!
Cheers!
Using old APS solution or very old APS powder for making APS solution can cause the leakage. Also overloading of protein samples can lead to the leakage. SDS PAGE wells have a particular loading capacity depending upon the size of the well.
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