problem in centrifuge of protein - (Sep/21/2007 )

hi
I`m making 1- centrifuge the meat tissue hemogenate at 1400 rpm
2- then recentrifuge at 15000 rpm for 15 minutes under cooling
3-then put 40 micro of supernatent + 10 micro loading buffer
4-then denaturate at 95 c for 5 minute
5-then centrifuge at 15000 rpm under cooling
every time I make this step
but now I make the step number 2 but not under cooling
then at the end of work the bands of protein is very weak
so I`m asking is not cooling desruct the protein
also I ask can I neglect the step number 5 that mean put the supernatent+loading buffer and denaturate only then inject it in the well
please reply me quickly to repeat the work
thanks


also

can I use stock of 1x running buffer but used 3 times before for 4 days
because I don`t have new stock

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persistent cooling should save nature of proteins and limit protease activity; loss of protein may be an effect of proteolysis; try to use, beside cooling, protease inhibitor cocktail

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Why do you want to skip the step 5? After you boil the mixture, you need to spin it down. We usually vortex either before boiling (just after adding in sample buffer), or after boiling, to make sure that things are mixed properly, then a brief spin (10sec or so is enough for us) to collect everything.

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we never reuse running buffer. most sds page protocols use discontinuous buffer systems so the buffers are not the same at the end of a run as they are at the beginning of the run. also, results may be less reproducible.

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