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wobbly bands in western blotting - (Oct/20/2008 )

Hi,

I get wobbly bands in my western blots. I equilibriate the gel for 45 min in buffer and do all the necessary wash steps.

Can somebody please explain why is it happening and the solution for it?

-cupidstunt-

You have to explain your problem in more detail: Since when are your bands wobbly and how does this exactly look like (picture?). Did you get always these results or is it a new finding? Give your full protocoll, which gels do you run, buffers, times, voltage, otherwise it is just wild guessing from our side here...

-biomaus-

QUOTE (biomaus @ Oct 20 2008, 10:50 AM)
You have to explain your problem in more detail: Since when are your bands wobbly and how does this exactly look like (picture?). Did you get always these results or is it a new finding? Give your full protocoll, which gels do you run, buffers, times, voltage, otherwise it is just wild guessing from our side here...

Hi,
Thanks. I have recently started WB after a long time. Earlier I used to get good bands, atleast not wobbly. I used 12% gel which is recomended for my protein of interesest. The recipe is inventoried in our lab and we use it with good results. Once the gel is set, loaded, I run it for 45min at 200v. Transfer for 40 min at 100v. Block in 5% milk solution in 1xtbs .1% tween for 40 mins. Then, primary in 5% milk overnight followed by 3 washes with 1x tbs -0.1% tween10. incubate with secondary for an hour in 1%milk solution. Followed by 3 washes with 1xtbs tween. incubation with avidin biotin 10 min each with 3 washes each. 20 min incubation with abc complex. followed by 2 washes with 1xtbs and 3 washes of d/w.

-cupidstunt-

O.k., I think, you should check if your gel is overheating. 200 V seems to be very high, the same is true for the transfer. I have used 35 mA and 55 mA for 2 mini-gels (stacking /resolving gel) for electrophoresis and 90 mA at a constant of max. 25 V for transfer (semi-dry). So, if your gel is heating up, this would be an explenation for the bands.

Try to run your gels in the cold room, if you won't reduce the voltage, this should help immediately.

-biomaus-

QUOTE (biomaus @ Oct 21 2008, 10:19 AM)
O.k., I think, you should check if your gel is overheating. 200 V seems to be very high, the same is true for the transfer. I have used 35 mA and 55 mA for 2 mini-gels (stacking /resolving gel) for electrophoresis and 90 mA at a constant of max. 25 V for transfer (semi-dry). So, if your gel is heating up, this would be an explenation for the bands.

Try to run your gels in the cold room, if you won't reduce the voltage, this should help immediately.

Thanks a lot. I will try reducing the voltage and also make up a new buffer.

thanks a lot

-cupidstunt-

Try putting the apparatus in an ice bucket while it runs.

-molgen-