How many O.D. cells to load onto SDS-PAGE? - (Nov/30/2008 )

Beginner question here:

I'm doing a analytical time course for a pilot E. coli recombinant protein expression experiment, and I was wondering how many OD600 of cells I should take as aliquots to analyze on the SDS-PAGE? This is small scale (maybe taking ~ 500 uL - 2 mL aliquots), and I'll be boiling the pellets to release the proteins. I want to keep the OD consistent so that the lanes will be easier to compare, but also not over/underload the protein gel.

Thank you so much!!!

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Sadly, this is one of the most time-consuming processes in science: standardizing.

I'd grow your bugs to the same OD (if you are looking for protein expression, maybe at least OD600 = 0.8), pellet and resuspend in the same amount of buffer, and load a gradient of volumes. After staining the gel, you'll be able to tell what amount to use in the future for clear bands.

For a starting point though, in my recombinant protein expression experiments, I take a 1mL control at OD600=0.6 or so, spin down, and resuspend in 100uL and run 10uL on a 12% gel. I don't know if that will work for you or not, but it might give you some place to start. Definitely run several volumes to check your own system.

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thank you so much! i pelleted all my samples to 1 OD and then resuspended in 100 uL loading buffer and loaded 10 uL of that and that seemed to be plenty.

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