I transformed my construct to yeast cells then i labeled my construct inside the cells. Now, I want to prepare lysate from yeast cells to extract the labeled proteins. I prepared spheroplast by lyticase enzyme but i can not completely lysis all spheroplast cells by sonification ( 1 min/ml) and extraction buffer. Extraction buffer contains 1% Triton x-100, 2 mM DTT and protease inhibitor.
Due to the low amount of labeled protein inside the cells i need to disturb most of the cells to get higher concentration of labeled protein in lysate.
Do you have any suggestion for that?
Hi Naghmeh,
In our lab we commonly use homogenization technique to extract proteins/mitochondria from yeast cells following zymolyase/lyticase spheroplasting, which gives us good yields (even though i cannot assure lysis of 100% cells). We wash the cells in 1.2 M Sorbitol and then resuspend the pellet in Homogenization buffer which contains Sorbitol, Tris-HCl, Protease inhibitors and BSA. Homogenization is carried out manually in glass dounce-homogenizers.
I can share details if you think this method may be useful for your purpose.
Thanks Madhuja,
Please let me know about details.
I am intrested to do in that way.
better is 2% SDS for spheroplast in sorbitol for yeast cells of any genera.
Hi Naghmeh,
The homogenization-lysis protocol is as follows:
1. Wash the cell pellet by gently resuspending (with pipette) in 1.2 M Sorbitol solution, before zymolyase treatment and spinning at 5000rpm for 10 mins.
2. For zymolyase treatement, we usually use 2 mg Zymolyase powder per gram of cell pellet (The buffer volume is usually 1ml per 0.15 gm of cell pellet ), at 30 degrees for an hour in a shaking water bath at a max speed of 65 rpm. Spin down the cells at 3000rpm for 5-6 mins.
3. Repeat Step 1 after zymolyase treatment step.
4. For homogenization, resuspend the washed cell pellet in buffer (1ml buffer per 0.5 gm of cell pellet) of the following composition:
0.6 M Sorbitol, 10 mM Tris-Hcl pH 7.4, 0.3% fatty acid free BSA and 100 mM PMSF.
5. Pour the cell suspension into a pre-chilled douncer, and use upto 30 strokes in ice manually (15 strokes - gap of 2-3 mins - 15strokes), following which you can transfer to centrifuge tube and spin down cells at 400rpm for 5 mins to obtain the lysate.
6. If required, the pellet can resuspended once again and steps 4-5 repeated, for maximum lysis.
Hope this helps....
Hi Naghmeh,
If you can't completely lyse your cells, most likely it means that your lyticase treatment is not efficient. I would use zymolyase instead, unfortunately it is much more expensive but it is a better enzyme to use. If you still want to use lyticase, you need to check the units of activity that the company providing lyticase states on the label, you might need to increase the amount you add. If you look at the existing protocols, you actually need to add a lot of it! I would also check the efficiency of spheroplast formation every 10 min and if necessary wait much longer for the process to be completed. The efficiency can be checked under the microscope, otherwise you can monitor the decrease of OD at 600nm (some protocols say 800 or even 900nm). For that you need to add you sample to water or 1%SDS which will cause spheroplasts but not cells to open. I hope this helps.
Regards,
Irina
Hello everyone,
I would like to know if it is posible to storage yeast culture/pellet before starting the spheroplasting procedure. Frozen, or in refrigerator and for how long.
Thank you very much.
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