regarding protein purification using 8m urea and sds page - (Apr/19/2014 )
i am trying to express plant virus protease in bacterial expression system (prset a b c, invitrogen).
up to cloning it is ok but when i proceed for purification using 8m urea in all buffers (as per Genetix Ni NTA system) under denaturing conditions i didnt get band on sds page.
i didnt quantified it.
i used to boil the purified protein sample by adding loading dye but i cant see band on sds page.
what care should i taken during sample preparation for sds page?
did you remove the urea before boiling? you can remove urea from the gel sample by drop dialysis (you don't have to worry about proper renaturing since you will be denaturing with sds sample buffer).
since the sample is already denatured, just add the sds sample buffer and apply to the gel, boiling not necessary.
keep in mind that the protein concentration may be very low so you may have to use a larger volume of sample/well or concentrate the sample.
you can silver stain the gel to visualize lower amounts of protein.
Do you know your protein goes into inclusion bodies or otherwise requires urea to be purified?
If you don't have a specific reason, then no need to make your life more difficult. Just purify it with normal buffer.
To be sure your protein is expressing, solubilize whole cells in SDS and blot for your his tag. Run side by side with a his-tagged protein that is sure to express.