Hi everybody,
I'm currently expressing proteins in and extracting from bacteria. The procedure is that I pellet the bacteria from bioreactor, resuspend in buffer (50 mM Tris/HCl, pH 8.0; 1M NaCl; 5% glycerol; 1 mM EDTA), pellet and once again.
Then I add the buffer again approx. in ratio 1:1 to the cells. And resuspend and then I lyse them with BioLogics model 3000MP ultrasonic homogenizer (with probe) with the following setting: 49 % intensity; 6 s ON; 9 s OFF for 5 minutes in total.
Because I usually have > 200 ml of the cell suspension, I lyse ~25ml at once, colled the lysed cells together and then go two more rounds; i.e. I lyse it three times, but not at once.
Then I pellet cell debris (and obviously undisrupted cells) and take away the supernatant.
The problem is, that when I resuspend these pellets again and lyse them once again (well, three times), I recover additional activity.
So my two questions are:
1) Does the cell density affect the efficiency of the cell lysis by ultrasound? If I diluted the cells more, would be the lysis more efficient?
2) is the cell lysis affected by the volume I use? I was told not to lyse more than 30 ml at once. I found an article, but there they had sonic bath and the volume was in range 100-700 ul.
Thank you
Hi Tomáš,
Both cell density and volume will affect the efficiency of cell lysis by sonication.
With a probe sonicator (as you are using) the wave pulses start from the tip of the probe and travel outward, losing power the further the go. so with larger volumes more cells are at a larger distance from the probe, reducing lysis efficiency. (this effect is less in a sonic bath but there the intensity is already reduced due to the waves having to travel though the bath to the sample)
The rate at which the wave pulses lose their intensity is affected by the viscosity of your sample (the higher the viscosity the faster the waves lose their intensity)
since having more cells in your suspension will increase the viscosity you thereby reduce the lysis efficiency.
one other factor is the tip size of the probe, smaller tips will generate more powerfull sonic waves. (but don't go over the maximum intensity indicated for the probe)
best regards,
Rob
I agree with Rob Masmen. Two more points to note:
Better you take optical density before lysis and after lysis. More than 80 % cells should be lysed for harnessing all the active protein from the cells. You can dilute the cell pellet like (10 ml lysis buffer/1g of the cell pellet).
I agree with both previous answers. I would like to add that your sanitation efficiency is likely to go up once you dilute your cells more (not 1:1, but more like 1:3). I have had great results at 1:3-4 with a maximum volume of up to 40mL. Also, adding DNAse to your lysis mix helps in my line of work with obtaining purer, less viscous samples.
Thanks for all the advices.
Well, I was trying to be as efficient as possible and thus trying to keep the total volume low, because it already takes so long. I will dilute it more now. Thank you again.
Hi Hi Tomáš! In my humble opinion u better add buffer in ratio 10-20 ml of buffer to 1 g of freezed pelet cells.
best regards,
Egor
I agree with the above comments, trying to keep volume low and density high is a false economy. You might try a couple/few rounds of freeze thaw before you sonicate the diluted samples too (if you have time). Depends on the bacteria but if this is a regular procedure it may be worth a check to see if it helps. I used to do this with Salmonella before bead beating them.
Hi,
yes, I actually freeze and thaw the samples usually few times, because it's usually few days/weeks between the fermentation and cell lysis and I see quite a difference between samples where I had time for these freeze-thaw cycles and where I did not.
However, as we brought up this topic - what is your protocol for freeze-thawing? The logic dictates to let it freeze slowly, so the ice crystals can form and grow thus disrupting the cells. But all the protocols I found use either dry ice or even liquid nitrogen. But liquid nitrogen one uses when one wants to preserve the cells, right?
Out of curiosity, how much (in volume) is approx. 1 g of cells? Because I do not weight them. Maybe I'll try tomorrow :)
I have an additional question, as Rob Mesman mentioned, that the sonic pulse goes from the tip of the probe.
I've always put the probe little bit above the bottom of the flask, to maximize the contact of the probe with the suspension. However, if it comes only from the tip, would it be better, to merge it only slightly?
Although the sonic waves go probably in all directions, so something in the middle would be the best? Stupid question, I know, but...
@Tomas Hluska Sonication technique may be important. I usually move my probe around (either up and down or in circles, avoiding the walls of my tube) while submerged completely to lyse all the cells. If the sonicator is nearly silent, your technique is correct. I sonicate in a very small volume (2-3mL of PBS/pellet from 250mL culture in a 5mL glass tube) and tend to get 1-8 mg of protein (depending on the fusion).
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