loading protein onto SDS-PAGE gel - (Mar/26/2006 )
Hi,
I expressed some membrane proteins and added deionized water and SDS loading dye to my samples. Then I heated at 95oC for 10 minutes, vortexed and centrifuged at 10 000 rpm for 5 minutes. I wasn't able to get any pellet so I loaded everything. When I loaded my samples onto the gel, I found that my uninduced samples were stickier than my induced samples. Within the same tube, I had a mixture of watery and sticky sample.
Anyone has any idea why this is so? Is my protein concentration too high?
Thank you.
-brainy-
QUOTE (brainy @ Mar 26 2006, 10:51 PM)
Hi,
I expressed some membrane proteins and added deionized water and SDS loading dye to my samples. Then I heated at 95oC for 10 minutes, vortexed and centrifuged at 10 000 rpm for 5 minutes. I wasn't able to get any pellet so I loaded everything. When I loaded my samples onto the gel, I found that my uninduced samples were stickier than my induced samples. Within the same tube, I had a mixture of watery and sticky sample.
Anyone has any idea why this is so? Is my protein concentration too high?
Thank you.
I expressed some membrane proteins and added deionized water and SDS loading dye to my samples. Then I heated at 95oC for 10 minutes, vortexed and centrifuged at 10 000 rpm for 5 minutes. I wasn't able to get any pellet so I loaded everything. When I loaded my samples onto the gel, I found that my uninduced samples were stickier than my induced samples. Within the same tube, I had a mixture of watery and sticky sample.
Anyone has any idea why this is so? Is my protein concentration too high?
Thank you.
what is your SDS loading buffer concentration? 1X?...i had same problem and i increased to 5X....and very important prepare new SDS loading buffer, dont use some left over in your lab....
-Kathy-
I added equal amount of 2X SDS PAGE loading buffer and water so that my SDS PAGE loading buffer is reduced to one time. Then I heated my samples to lyse my cells.
-brainy-