Hi Everyone,
I am going to purify a GST tagged protein from bacteria. I don't have a fixed angle rotor in the lab at the moment to centrifuge the samples after lysing the bacteria. I only have swinging bucket rotors and the max speed I can go is 4700 rpm (4816 g). Has anyone tried to use a swinging bucket to centrifuge bacterial cell lysates?
Hello Engin,
Yes you can. Centrifuge at 4500 x g for 30 min. Check if you obtain clear supernatant. If not, spin for another 15-30 min.
Hi Syed,
Thank you very much for your reply. That's what I was planning to do but with that low speed I wasn't sure if I could pellet all the 'junk'. Doesn't the supernatant become more dense or viscous with low speed centrifugation? Were you able to purify your protein successfully?
Relatively low speed can be compensated (to some extent) with longer runs. Ideally, the bacterial lysates should be centrifuged above 15,000 x g for 10 - 15 min but if it is not possible, you can spin for longer times and see if gets the job done. We have fixed angle rotors so never happened to spin cell lysates in swing rotor buckets. Most cell debris will come down but I am concerned about insoluble protein aggregates. Since you have no option, just give it a try.
Hi Fu,
Thank you for your answer. Can you please elaborate it a little bit? Why do you not suggest it? What do you think might happen? Have you tried it before and didn't it work?
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques