I've had the best success by digesting the cell wall with Zymolyase and then disrupting the resulting spheroplasts with a tight fitting Dounce homogeniser or mild sonication on ice. I then add Triton X-100 to a final concentration of 0.1% and mix at 4 degrees for 30 min. I routinely use this procedure to generates lysates from which I isolate proteins by affinity chromatography. This procedure will take 4-5 h. The method I use is in the attachement

Bear in mind that the large-scale PPI by MS papers used beads for lysis, so you can recover complexes from lysates generated in this way. However, in my hands, beads just aren't that good.

Hello:

Breaking yeasts is easy using glass beads (Ballotini, SIGMA). It can be done manually in a vortex or using a Fast-prep machine. If done manually you should use glass tubes. Keep temperature as low as you can by maintaining samples on ice between breaking pulses. There is plenty of literature about the method.

It is faster and cleaner than spheroplasting in our hands, and suitable for enzymatic assays.

Cesar

You can use from Glass beads for griding and then phenol choloroform

Vortexing with glass beads as suggested by Cesar is a cheap and efficient way for cell lysis. We usually disrupt cells in microcentrifuge tubes. For efficient cell lysis use 2ml microcentrifuge tubes or other tubes with near flat bottom (in 1.5 ml tubes with sharp conic bottom the lysis is much less efficient). To avoid heating the samples, disrupt cell during 30-60 sec with 20-30 sec pauses on ice. The total time of 3 min is sufficient for efficient cell lysis. To avoid, protein denaturation on the foam, add 1/50 volume of isoamyl alcohol, it inhibits the foam formation and do not interfere with protein-protein interactions.

As Cesar and Dmitry have mentioned, glass bead lysis works pretty well. I have used that method to generate cell extracts that retain translational activity and we have done affinity purification from those kinds of lysates as well. I found that glass tubes work well - lysis is more efficient, requiring less cycles of vortexing, and thus less probablity of dentauring your protein of interest. What kind of protein-protein interaction studies are you doing? Co-expressing your proteins of interest or mixing lysates together?

I have one method that allows me to obtain about 300microL of whole cells protein extracts with the protein concentration about 10-30 mg/ml.

Protocol for 100mg of cells:

1.Cetrifuge colony and wash twice with distilled water.

2. If you have for example 100mg of pellet - resuspend the pellet with 150ul RIPA buffer (add 1mM PMSF before resuspension). Then add 100mg of glass beads (0,25-0,5mm).

3. Vortex in eppendorf tube 1,5ml 1 min. followed by 1 min. pause on ice (cooling). Repeat it 4 times.

4. Centrifuge the tube (everything, also with glass beads) at 2 000xg (4oC). Then collect supernatant (you can use pipette with yellow tip glass beads will not get across).

5. Add 150ul fresh RIPA buffer and 1mM PMSF into pellet (with glass beads used before) and repeat step 3 and 4.

6. Combine supernatants (the protein concentration from first vortex will be higher).

7. Centrifuge at 14 000xg and discard pellet.

You could use boiling or glass beads. In the first method, cells are suspended in distilled water tubes and incubated in a boiling water-bath for 5 min. In the second, you can vigorously vortex cell suspension in tubes cointaining sterile glass beads.

I use sterile glass beads for cell lysis. It works very well

I was "successful" in using MHz ultrasound (= no cavitation, hence no free radicals) when Ethanol was present in a very low concentration (3-4%). See my paper in Bioseparation or Ultrasonics (both 2000)

I've routinely used two methods that both work well: (1) freeze-fracture by moving from liquid nitrogen (or an ethanol/dry ice bath) to hot water, just long enough to thaw, then freezing. Repeat many times (8-10). Works best if you are using a very protective buffer like Laemmli. The benefit is it is pretty simple and the yield is reasonable. (2) Sonicating with very small (like 10um) glass beads. This is similar to the suggested vortexing with glass beads or sonicating alone but much much more effective. This method is by far the best I've ever used, I've never been able to achieve comparable yields with any other method. Make sure you sonicate in bursts and keep the sample on ice to avoid heating or damage to your sample.

Jacques Thierie

I don't have a lot of information, but I also succeeded to break S. cerevisiae by using a simple ultrasound cuve. The "kinetics" trhough the microscope (in visible light) show clearly an homogeneization od the cell suspension. More examination is necessary to correctly evaluate the results.

Glass bead lysis is fast and can be efficient, but lysis of spheroplasts in a Dounce homogenizer, as suggested by James Matthews, is good also, especially for larger volumes. If looking for membrane proteins among your interacting proteins, be careful how fast you spin your initial extract to clarify.

I have some experience in lysis of yeast Pichia pastoris. Results after enzymatic digestion or vortexing with glass beads did not help for complete cell lysis. For my purpose, grinding with liquid nitrogen worked better and I recommend it if it suits your purpose. I guess proteins will maintain its native structure in N2. Please clarify that and proceed. Ready made lysis solutions also may give good results.

This method was effective:

Yeast strains were gown for 24 h in YPD medium containing 5 % glucose at 30°C. The cells (20 ml suspension) were washed twice with ice-cold sterile distilled water and centrifuged at 4000 rpm for 10 min at 4°C.Yeast cells (100 mg, dry weight) were re-suspended in 1 ml buffer solution (prepared as described below) at 4°C and then separated by centrifugation (4000 rpm). Each cell pellet was then transferred to a 5 ml flat-bottomscrew capped test tube containing 1.5 g of glass beads (0.5 mm diameter). The buffer solutions employed for the extractions were:

• 100 mM Sodium Acetate Buffer, pH 5.5 for, containing 1mM dithiothreitol and 20% glycerol (w/v) for the maltase assays

• 100 mM Sodium Acetate Buffer, pH 4.6 for invertase assays

Cells were disrupted in a vortex shaker for four periods of 30 sec with 1 min cooling intervals on ice. Duplicates of disrupted cells were quickly centrifuged (3 min at 12,000 rpm) and the supernatants assayed

All the methods mentioned above by other researchers are efficient under circumstances and each method can be aplied for secific puroses- Depends what we want to isolate from the yeast cells. For example, grinding with glass beads is good for isolation of soluble cytoplasmic proteins or the cell walls but not for preparation of intact mitochondria or plasma membranes. For latter purposes, enzymatic lysis or gentle protoplast lysis are more appropriate. In some cases, osmotic shock can permeabilize yeast cells thereby allowing the"in situ" determination of enzyme activity (see e. g. Sestak and Farkas:.Anal.Biochem. 292: 34-39 (2001).

Dear colleage, you may use a glass beads (about 1/3 of cells pellet) and shaker (very fast regime). This procedure should be controlled by microscopy. About 10-20 min need to breakage about 80% of cells.

Ideal is use of cell wall lytic enzymes followed by mild grinding of the resulting with acid washed glass beads using a protective buffer or subjecting the resulting sphaeroplasts to an osmotic shock.

I always use glass beads, as someone said previously. You can fill your sample with an amount of glass beads proportional to your pellet, then add a buffer (TCA 5% or a breaking buffer containing an anti proteolytic enzyme) and vortex the sample for about 10 min. Which kind of assay do you want to perform?

Zymolyase + homogenizer (as James suggested) will give you cell lysate for studying the soluble enzymes and the plasma membrane fraction to study membrane proteins. Also you can try to permeabilize the cells (0.8M sorbitol + 0.05% Trton X-100 and freeze at -70C overnigth) and measure activity in situ.

I had very good results with 2 MHz ultrasound having a few % EtOH in the supernatant. I am aware, that such ultrasonic fields are rare, but maybe you find an US device in the tens of kHz. Common knowledge is, that yeast is hard to break, but maybe results are better when adding a little EtOH.

Best regards, Stefan

PS: Would be interested to hear about the outcome btw 

 b

Hi,

You can use the freeze thaw or glass beads.

Best,

Zarrinfar

Dear James,

I am also looking for a better protocol for lysing yeast cells without harsh lysis using glass beads as it may result in loosing some interacting partners. The protocol which you have suggested i.e using zymolyase treatment and lysing the cells with homogenization. But I have a doubt would it be a whole cell lysate covering both the cytosolic and nuclear proteins. I will be grateful to you if you can help me with this.

Thanking You

Vivek 

Vivek,

You are probably right, that protocol might not completely disrupt the nuclear membrane. I think you have to determine whether you really need a total cell lysate or whether it is a better idea to generate cell fractions. I'm sure adding some digitonin and/or octyldecyl glucoside would help disrupt the nuclear envelope but then you have to wonder how these detergents might effect protein-protein interactions.

What is your application?

Use LiAc and NaOH to disrupt cells. It will not affect protein-protein interaction and activity of protein. 

For reference please find the attached paper.

Dmitry S Karpov can you give me that cesar's paper?