SDS-PAGE - Trouble in running SDS-PAGE (Jun/02/2002 )
I have now running the SDS-PAGE for determining Rotavirus for my thesis and got a trouble. Using the 10% resolving gel and 3.5% stacking gel, but my bands were not sharp, they were blur and sometimes distorted. When I made the resolving gel and overlaid by distill water (about 500-600ul), after the gel got hard, I found a thin layer of water at the bottom of the hard gel. One more thing is sometimes my stacking gel could not be getting hard enough; it left a little bit water after got hard.
Is there anyone known about my problems, please help me. Thanks very much in advance. Here is my email address: [firstname.lastname@example.org.]email@example.com.[/email]
Blur and distorted bands may come from a large amount of your protein. Usually, I use 5% stacking and 13% separating gel without any problem. After adding APS and TEMED, you must pour fast, in order to get well polimerized gel.
Thanks very much for your answer. But the problem is I only use 16ul sample and 4 ul for loading buffer. Even though with RNA extracted from SA11 ( from cell culture)by phenol/chloroform method, I also got these matters. By the way, how many voltage( or Ampere) do you usually use for running the gel and how many hours?
Hopefully receiving your suggestions soon,
I also generally use 10% resolvin and 3-3.5% stacking gel, but usually don't get such problems. anyway, you can check with your %-age of APS, and try a little more amount of APS to prepare your stacking gel. it does not harm the protein sample.
regarding your resolving gel, do you use glycerol to prepare this gel? approx. 10% glycerol in the resolving gel solution is good for the clarity. after pouring the resolving gel you may not need to overlay it with water, instead you can slowly add the stacking gel soln. over it directly with a pasteur pipette,but when pouring it you must move the pasteur pipette to and fro along the length of the gel. this procedure is good for avoiding the extra water in the gel, but need a practised hand.
another thing you can check, the preperation of the acrylamide soln. should be correctly 30%, if there is any mistake the final stacking gel as well as the resolving gel concentrations will be changed, and may be that as the %age of stacking gel is low it is not properly getting hard, while the resolving gel though being ultimately less than 10% , getting hard. you should check it from the size of the protein marker in the gel. usually you should view upto 20kd proteins in 10% gel.
I recommend to run the gel in constant volt of 200V in a BIO-RAD minigel apparatus.
i have been working lately with west nile virus and have no trouble at all with the sds.form what u say i could understand that the amount of temed and aps may not be sufficient to have ur gel polymerised completely.
also u can check ur acrylamide which when contaminated may give u improper staining and poor polymerization.i have no idea of how water can saty at the bottom when ur gel hardens...watre is lightere than ur gel and will float.if u have trouble then try using ethanol but should really matter.check ur gel recepie...if u have trouble get the lamenellis sds gel receipe...it works just fine.try using a 12% gel...works with great results for me
the gel u can run at 50 volts until it runs through the stacking and then run at 150 volts tilll about last couple of centimeters of the gel.if u dont run ur gel right u may end up with distorted bands.
The thing to make sure if your gel isn't polymerizing well is
1) that your 10% APS is not old. You should make fresh APS every week or two.
2) that you add enough TEMED.
3) that you are waiting long enough (don't disturb the gel for at least 30 min after pouring the mixture).
i think the problem maybe:
1. the period is too long to stainning it with comassie blue after you run gel;
2. the protein was loaded more than the gel resolution on the short distance.
i know this is going against popular opinion but try running ur gel at a fixed current instead of voltage. a good try would be 15 mA to 20 mA depending on the size of gel. if u are able to run it at cold temperature, i.e. cold running buffer in a cold room or fridge.
for my case this greatly increased band intensity, especially the smaller ones that migrates close to the dye which usually diffuses.
there are logical reasons for this, i think u can look it up in textbooks.
hope that helped.
& don't forget to de-gas your acrylamide gel mix under vacuum (before you add TEMED & APS) for ~30min.
Try to overlay with Isopropanol for example instead of water it also may help to get a clear separation line bettwen the Stacking gel and the running gel, wath may also improve your run.