I am having trouble getting enough protein to run SDS-PAGE, I barely get 50-60 ug total protein as my total concentration in a final volume of 150 ul.
I grow my tumoursphere in Corning ULA 6 well plate for around 7-10 days with an initial seeding density of 5000 cells, tried 10 000 cells but I get more uniform spheres and lesser aggregates with the 5000 cells seeding density. I lysed the cells in 1% SDS pre-heated to 95 degrees, homogenised using 20g needle 10 times and centrifuged at 12000xg at 4 degrees.
I guess one way is to do in more replicates then pool all the wells together, but the plates are quite costly to keep doing that. Does anyone want to suggest other (perhaps more economical) ways?
Try to optimise your lysis protocol, try to use RIPA buffer or M2 lysis buffer instead of just SDS with amount of 50-75 ul per well. Maybe you can also add sonication I did not work with tumorosheres but have an impression that they more robust that usual cells.
hello zubaidah, it appears that you are loading around 7-8 ug of protein on SDS GEL. It must showup there. Make sure you are processing the sample well and try to use fresh loading dye. In addition to that the amount of soluble protein can be increase by using appropriate cocktail of chemicals as suggested by Arvind and Anton.
Good luck..
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