Unfortunately, I had to stope expression process for about 5 months and after this delay time I had to again to do some change in expression conditions I mean against before I used 25◦C to participate bacterial suspension. But after purification step, there was no band in SDS PAGE. I don’t know what is the wrong? Is this possible that this change in centrifugation temperature caused I lost my protein? My protein is HIV-1 Tat and Expression temperature is 37◦C for 4 hours and I use BL21DE3 and PET28(a).
do you have any idea? Please?
Dear Zahra
Considering that bacteria are grown at temperature as 37/30/25°C (in your case 37(i in generally there is no main differences if you centrifuge the bacterial pellet at 25°C or 4°C. (The important is that you do not reach higher temperatures that can lyse the bacteria)
Generally what is more critical is the temperature of the centrifugation that you use after cell lysis, because in this case the protein is released from cells, can become in contact with proteases and is it better that in the time that occours beetween lysis and purification (that need to be the shorter possible) the sample need to stay at low temperature.
Did you can supply me more details? Do you monitored the protein expression and solubility in SDS-page before purification to see where it is lost?
ciao
Manuele
Hi Zahra,
I second Manuele, it should be not a big problem to use 25C instead of 4C. Bacterial suspensions need only little time to sediment in centrifuges, 5-10 min at 4000 g should be sufficient in most cases. Therefore centrifugation at room temperature should be fine - however, uncooled centrifuges heat up quite a lot during the centrifugations (i.e. when used for several times in a row) and rotors can easily reach >50C (which would be a problem for your cell suspensions...).
Next time take an aliquot of the unprocessed culture, spin it and resuspend it in one part 8 M urea and one part 2X Laemmli buffer, heat and check by SDS-PAGE. That will give you an idea of whether the culture failed to express the protein or something went wrong during purification.
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