Protein was expressed in SHuffle cells in presence of 0.5%glycerol in the LB media(230ml).
pellet was dissolved in 20ml lysis buffer(50mM Tris pH 8.0, 150mM Nacl, 10% glycerol).
i couldnt see any difference to resuspended pellet after applying sonication to the pellet.
(20 mins, 20 % amp, 30s ON, 30s OFF). then i further extended the time to 10 mins i saw little difference in the solution. Then again i extended the time about 10 mins.
In the purification, most of the protein went in pellet. I doubt that cell lysis process is not done effectively or its too much sonication.
-
Before further repeat this experiment, what are the things we can tweak?
Hi,
Your protein is going into inclusion bodies where you cant see your protein in the supernatant even after proper sonication.
Try to reduce the induction or tempearture where you can get the protein expression which is easily to come in supernatant. You can also add mild detergent before sonication and check for the protein in the supernatant.
You can use Triton X100 and EDTA at 0.1% and 5 mM conc, try sonication till the protein content in the supernatant/ cell lysate becomes constant.
Now considering that it failed, I will take a tight control of an experiment.
I will dissolve pallet in 60 ml lysis buffer, divide it into 6 different vials
1) One I will follow what you just did
2) I will double the sonication time
3) I will decrease it by half
4) I will do 3 freeze thaw cycle
5) I will add very low percentage of detergent and rotate it for an hou/30min at 4C and then sonicate
6) I will do nothing (Control)
In this way, I can make an approximate guess of what might work in my case.
Considering what you wrote in lysis buffer. If you missed to add protease inhibitors then please dont waste your precious thought on this prep.
You may try 16 to 20 C
My experience is that bugs are lysed effectively in about 10 seconds using a probe sonicator: a sonicator bath (well, the one we have) does nothing. But there are many different probe sonicators, so my suggestion would be to check the integrity of your bacteria by microscopy (which is what we did in our original studies). If you dont get a high frequency of lysis, continue sonicating, or try a different machine (our machine gives a +/- 23oC increase in temperature with 1mL of buffer in a 1.5mL Eppendorf with setting 80% and one pulse of 10 seconds). If you do have extensive lysis, but your protein is still in the pellet, then you have inclusion bodies, and you can act accordingly.
Thank you all !
Kannan Balakrishnan, Mahesh Chandak , Shamsher S. Kanwar Geoff Margison
I will experiment with your suggestions.
Mahesh Chandak :
Ya, i didnt add any protease inhibitor. In subsequent prep i will add.
I know that they protect the protean of interest from protease. Is protease inhibitor plays role in lysis of cells?
Depending on cell type you will have serine, cysteine, aspartic, aminopeptidases, metalloprotease or thermolysin-like activities and when you lyse cells, those proteases will digest your protein of interest. In order to prevent this you definitely need protease inhibitors when you start protein purification prep- Usually 1X or higher concentration of cocktail tablet and metalloprotease are preferred as long as your protein is not metallosensitive.
Some protease are cell permeable some are not.
I am guessing 10% glycerol at the time of lysis might be creating difficulty in checking the lysis. I think you need to change sonication strategy. Following are some tips
1. Check the color of cells before and after lysis. The lysate should not turn blackish, it means you have over lysed the cells
2. I prefer sonication for 1ltr culture cell pellet: 5min, 5sec on/off
3. You will observe change in consistancy/ fluidity of solution after lysis
4. If the pellet appears white, your protein might be simply going into inclusion bodies.
- Badges
- Science method