cell homogenization - (Jun/10/2011 )
Thank you Uncle Rhombus, I'll definitely try that.
-zienpiggie-
Rhombus comes through once again...
@yimaio - the clearance large and small is the maximum gap between the sides of the tube and the pestle used to homogenize the samples. With particular size gaps you can rupture the cytoplasm but not the nucleii for instance.
-bob1-
rhombus on Mon Jun 13 10:33:53 2011 said:
zienpiggie on Mon Jun 13 05:16:10 2011 said:
oh wait, you said snap-freeze right. so I have to basically be pretty quick. I dunk it and take it out in split seconds kind of thing. I think I should just go ahead and give it a shot.
Dear zienpiggie,
....
Sonicate the cells 3 times for 5 seconds a times using an optimised sonicating power/frequency....this is done on ice and there is a gap of 30 seconds between each sonication.
ultracentrifuge the disrupted cell suspension for 30 minutes at 105,000g
...
We have used this method many times and have published data to support the above:
Biochem and Biophys Res Commun. Volume 188 No. 1, Pages 209-215, 1992 Moncada et al Glucocorticoids do not affect the induction of a novel calcium-dependent Nitric Oxide Synthase in Rabbit Chondrocytes.
I do hope you find this useful.
Kindest regards.
Uncle Rhombus.
Hi Uncle Rhombus,
I hope I am not misunderstood your suggestion. BUT i still have to ask again, did you mean that the sonication just need 3 times with 5 seconds sonications and 30 seconds between each sonication? That means you just need 5*3+30*2=1minute and 15 seconds to finish sonication.
If I have 1L Hi5 cells, I usually use 100ml lysis buffer, dose such sonication time enough? PS: what is your proper power on your sonicater? Thanks
-rainlqy-
rainlqy on Wed Jul 6 07:54:43 2011 said:
rhombus on Mon Jun 13 10:33:53 2011 said:
zienpiggie on Mon Jun 13 05:16:10 2011 said:
oh wait, you said snap-freeze right. so I have to basically be pretty quick. I dunk it and take it out in split seconds kind of thing. I think I should just go ahead and give it a shot.
Dear zienpiggie,
....
Sonicate the cells 3 times for 5 seconds a times using an optimised sonicating power/frequency....this is done on ice and there is a gap of 30 seconds between each sonication.
ultracentrifuge the disrupted cell suspension for 30 minutes at 105,000g
...
We have used this method many times and have published data to support the above:
Biochem and Biophys Res Commun. Volume 188 No. 1, Pages 209-215, 1992 Moncada et al Glucocorticoids do not affect the induction of a novel calcium-dependent Nitric Oxide Synthase in Rabbit Chondrocytes.
I do hope you find this useful.
Kindest regards.
Uncle Rhombus.
Hi Uncle Rhombus,
I hope I am not misunderstood your suggestion. BUT i still have to ask again, did you mean that the sonication just need 3 times with 5 seconds sonications and 30 seconds between each sonication? That means you just need 5*3+30*2=1minute and 15 seconds to finish sonication.
If I have 1L Hi5 cells, I usually use 100ml lysis buffer, dose such sonication time enough? PS: what is your proper power on your sonicater? Thanks
No problem at all.....you have understood me perfectly:-
sonicate for 5 seconds (on ice) at an optimised power....then let the cell suspension rest on ice for 30 seconds.
Repeat this 2 more times.
Excessive sonication causes heat, and heat will denature your enzyme, thus inactivating it. You have to optimise cell fractionation i.e. release the protein from the cells...check the cell suspension under a light microscope to see that the cells have been fully disrupted.
On the MSE Soniprobe I use, the power rating is in Amptitude microns....and I use 50% of maximum sonication power.....again this level has to be optimised.
Different cells require different disruption powers......so lots of optimisation!!!!
Have fun and good luck.
Kindest regards.
Uncle Rhombus.
-rhombus-