I am using a self made Bis-Tris gel in combination with MOPS buffer. The attached picture shows a 1.5 mm comb with 10 wells. I am loading 20 µL of the sample and 8 µL of the protein ladder (10–235 kDa). The gel is run in Two rounds starting with 85v for 20min then at 100v for 1:20 min.
Any suggestions regarding gel preparation, sample loading, running conditions, washing, or imaging would be greatly appreciated.
Dear Ouzaouit Abdelhak
of course using a pre-cast gel with gradient you can improve the separation.
using your hand made gel probably you can little improve the quality of the image by:
- reducing the sample load to reduce the buckground; 10ul sample and 4ul ladder can be enough
- use the MES buffer
- Increasing the voltage (150 or 180V can be ok)
best regards
Manuele
You can try to
- use gradient gels
- differentiate (de-stain) the gel longer to reduce background
- reduce the amount of protein per lane
- use fluorescent staining, for example with RuBPS (dye hard, doi:10.1007/978-1-61779-821-4_48)
The previous comments are all good, I would add for a gel you make yourself, make sure it is fully polymerized. Try making 4 gels at a time and store overnight in the frig inside a light tight box before use. And then run the gel in a frig or cold-room, or with a cold pack in the buffer chamber. I always liked to make Tris glycine gels. Do you deionize your acrylamide? Do you store your acrylamide in the frig in a container wrapped in aluminum foil to protect if from light?
Thank you all for your suggestions I would definitely try those comments. Dear @Gary S.Laco for the Gel I always run strait after making sure that is completly polymerise without storing it overnight in the fridge. Deonizing the acrylamide from charges I’ll give it a chance too.
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