Ultrasonic homogenizer for protein and enzyme extraction? - (Nov/09/2006 )
Dear All,
Could anyone out there answer me these questions, please!
I want to extract and isolate the membrane proteins/enzymes from animal tissues. May I use the ultrsonic homogenizer to homogenize the tissues before applying differential centrifugation for isolation? Does it detroy the organells or denature the proteins? I knew that ultrasonic homogenizer produces lots of heat during operation. I homogenize the samples on ice in 2 minutes with a pause of 1 minute every 30 seconds. Is it enough to avoid protein denaturation? What kind of buffer is the best for this application? I have in hand several types of buffer but I am a bit confused to choose one. May I use the same buffer (TBS) as I used for total protein extraction?
Thanks a lots for your time and help!
Dear Minhhanh,
You should be OK. I have used an ultrasonic probe for homogenisation of cells and tissue in the past. I was after functional iNOS enzyme for drug screening purposes. The enzyme was fully functional after the following protocol :-
Cell suspension on ice
Sonicate at 10 Amplitude Microns for 5 seconds........rest on ice for 25 seconds
and repeat........rest on ice for 25 seconds
and repeat.
Resting in between sonications reduces the temperature rise and will therefore reduce the risk of DENATURATION.
I have not tried to look at organelle integrity, so I cannot help with that.
You will have to optimise your conditions for your cells/tissues.....and I don't know what sonicator you have either.
Hope this is useful
Rhombus
You should be OK. I have used an ultrasonic probe for homogenisation of cells and tissue in the past. I was after functional iNOS enzyme for drug screening purposes. The enzyme was fully functional after the following protocol :-
Cell suspension on ice
Sonicate at 10 Amplitude Microns for 5 seconds........rest on ice for 25 seconds
and repeat........rest on ice for 25 seconds
and repeat.
Resting in between sonications reduces the temperature rise and will therefore reduce the risk of DENATURATION.
I have not tried to look at organelle integrity, so I cannot help with that.
You will have to optimise your conditions for your cells/tissues.....and I don't know what sonicator you have either.
Hope this is useful
Rhombus
Hello Rhombus,
Thanks a lot for your message. Yes, of course it is useful for me:). I just have another question concerning "Amplitude Microns". How could you set it? Perhaps you have different sonicator than me. I have LABSONIC U from Braun with output power of 170 Watts per min after 1 min. It is quite old instrument I there have been no one here using it for long time, therefore I have some difficulties with technical problems. In your case, how long did you homogenize your sample (in total, not including resting time)?
Once again, thanks for your help!
Minhhanh
Hi,
I use sonicator to homogenize animal cell lines and then I isolate microsomes from the lysate. I use the following procedure:
4 to 5 bursts of 4-s duration, amplitude-30% and pulse-1
Hope it helps.