I have to break yeast in order to collect the proteins for purification purpose. I have started 3L of cultures thinking that I had a protocol, but I don't have one...
I have a french press in which one I can put 40mL of sample at a time. So basically, I will pellet my culture, resuspend the pellet in my purification buffer and pass the sample in the machine.
My question is: is there a limit of density at which the lysis will be less effective? If so (an I guess there is), what it is?
If you have some advice, I will be glad to hear them and to discuss with you.
Hello,
I don't have a turrax.
I have the second kind of french press you describe. There is one hose in which one I fill and empty the piston, but I can fill it as you describe. The total volume I can put is 40mL. I have put a picture of the model.
It is a very old one but it seems to work well. As nobody uses it in the lab, they can't help me in any way.
Yeast cells are resistant to mechanical shear. i recommend enzyme treatment (zymolase). Macerate in liquid nitrogen is one of the best strategy to lyse these smart ones..
Depends on your downstream application, but I had experience using microwave on high power (it will boil wildly, so use bigger container) for easy extraction.
I assume you are using S. cerevisiae? 1 gram of cell pellet per 1 mL of your lysis buffer is sufficient. Make sure you have the instrument set at the correct PSI for yeast. Do three passes and you should achieve >75% lysis. Make sure you control the flow very well to maintain the PSI necessary to lysis yeast. Do not microwave your sample if you plan to do any enzymology. Zymolase works but French press is faster. Make sure you chill the pressure cell in a cold room.
Hello all,
finally it worked well. So for those of you who are interested by the technic it is quite easy and worked very well.
-Start a culture of 1,5 L with YPD and put it at 30°C for 48H
-Pellet the culture
-After washing the pellet with water, resuspend it in 40mL of purification buffer (40mL is the final volume) added with 1mM of PMSF and cocktail of yeast protease inhibitors.
-Break the yeast at 20000psi, 3 passages is enough. Apparently it is useful to do at least 2 passages. As this kind of machine heat a lot it is better to put the piston in a cold room the day before and to work all the time in ice.
-Pellet the lysate 30 minutes at 30000G.
-Take the clear lysate for your stuff.
The fact is that when the yeast are broken they turn in a orange/pink colour instead of brown. I don't know where it comes from but it is helpful! It is true at least for K. Lactis.
So to answer
@ Ancy Thomas: I am afraid that lysing the yeast in liquid nitrogen may alter the structure of my proteins. As it is for purification and biochemistry assays, I won't try it.
@Yanuar Sulistio: for the same reasons as above (and even more!!) I will not try boiling them :D
Feel free to complete this protocol or give your feedbacks if you try it.
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