Dear all,
I have a question: when I used the silver plus staining kit from bio-rad, at 15th min of the staining step, I observed clear bands. Since the protocol suggested 20 min for staining, I stained for another 5 min. However after 20 min, all the gel become brown or black! The bands were not obvious any more. Does anybody know the reason?
Thank you!
Dear Kangzi,
We usually use the bio-rad kit and we don't have any problem. I would give you some advice it may help.
- Try to use a very clean Dish: sometimes it is necessary to wash the dishe as this=> Add 30ml (cover the bottom) with Nitric acid 8M to dish and leave it for 1-2 min, then rinse it by MQ water before starting.
- For Staining Step just keep watching the gel. In my case it takes just 10 min to see the bonds and after 15 min it would be completely brown.
- Add stopping buffer when you see the bands immediately.
Good Luck
Ladan
i agree with Ladan when you have a good result stop the reaction , Ignore the instructions which are generally good and fix the result when you like the result because you are over developing
I have not used the BioRad system, but have used the Morrissey protocol (Anal Biochem 117:307) a lot. As stated by Ladan clean surfaces is important. Both in the electrophoresis cell and the staining trays. I rewash the electrophoresis cell and plates just before using them. I clean and then silanize the glass trays I used. The quality of the water used is also very important. After all that, each gel is allowed to develop 'to taste' as optimizing the signal to background varies greatly from sample to sample. But, once you become familiar with the protocol you are using, its a great way to visualize low samples where the protein content is low.
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