SDS-PAGE Help

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wondervulva
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SDS-PAGE Help

Post by wondervulva » Wed Nov 08, 2006 1:00 pm

Hello,

By running SDS-PAGE analysis with NuPAGE gels and buffer, if the sample is boiled the protein precipitate and doesn’t enter the gel. Without boiling (or heating) on the other hand, the protein can’t be fully denaturated and therefore it migrates as several bands instead of the expected single band.

How can I solve this problem?

Thanks,

rasap
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Post by rasap » Wed Nov 08, 2006 1:52 pm

Hi,
I guess it depends also on the loading dye solution which you are using, this one should contain SDS and DTT, then you will not need to boil for so long.
Try Fermentas 5X Loading Buffer Pack and the protocol like here:
Thaw the pack’s components either at room temperature or at 37°C to dissolve precipitated
solids.
Vortex gently, but thoroughly to ensure that the solution is homogeneous.
Add the following into the screw-capped-tube:
– 2.5μl of 20X Reducing Agent;
– 10μl of 5X Loading Buffer;
– Protein sample;
– Water, nuclease-free (#R0581) to 50μl.
Incubate samples at 95-100°C for 5 minutes.
Spin for a few seconds in a microcentrifuge.
Apply directly on SDS-polyacrylamide gel.
http://www.fermentas.com/catalog/electr ... ufpack.htm

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