Sample preparation for SDS from detergent insoluble fraction (Pellet). - (Mar/12/2011 )

Hi everyone....
i wish to screen some proteins in the detergent insolule fraction - the pellet settles after protein clarification. if i have to run this as well, how shd i prepare the sample? my plan is to add the loading dye directly and denature after mechanical disruption thru syringe, will it pull down the protein or shd i have to prepare some stronger lysis buffer inorder to see the detergent insoluble proteins in the pellet??? Kindly shed your expertise or suggestions.

Thankings,
Gnana....

-GNANA-

if the pellet is not too large then you should be able to solubilize the proteins in sds sample buffer (with reducing agent) and heat (60-70C for 10-20 minutes or boil for ~5 minutes).

-mdfenko-

thank you....i do this and get back if i have any probs....thanks for ur reply.........

Gnana..

-GNANA-