We work with a two-hybrid system for studying the interaction of viral proteins and use for this purpose, the yeast strain Y 153. We have our antibodies to viral proteins, and want to detect their fusion with the DBD and AD domains Gal on Western blots. But we don’t have glass beads. Who knows the alternative methods for lysis of yeast cells?
The kushnirov protein extraction method (Yeast. 2000 16(9):857-60) is routinely used in my lab and works very well without the use of glass beads. NaOH and SDS page sample buffer are simply required to extract total proteins from cell amount equivalent to 2-3 OD600.
Good luck
The kushnirov protein extraction method (Yeast. 2000 16(9):857-60) is routinely used in my lab and works very well without the use of glass beads. NaOH and SDS page sample buffer are simply required to extract total proteins from cell amount equivalent to 2-3 OD600.
Good luck
You might have a look on the method by Tobias von der Haar (PLoS ONE, 2, e1078) http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0001078
We used this method only once to confirm expression of a bait protein for yeast two-hybrid screening and it worked right away (and in my recollection better than a glass bead lysis protocol we used before).
Good luck
We use routinely alkaline lysis to get total yeast protein extracts. It's just adding 150 µl alkaline lysis buffer (975 µl 2M NaOH + 75 µl beta-Mercaptoethanol) to 1 ml of cell suspension, mixing, incubating 10 min on ice, than precipitating the proteins by adding 150 µl 55% TCA, mxing, incubating at least 10 min on ice (at this point samples can also be stored at -20°C for later preparation) and finally pelleting the protein extracts with 13 rpm for 10 min and resolving the pellets in HU buffer. After 10 min denaturing at 65°C and a short centrifugation to get down the cell debris you can apply the desired amount of supernatant onto SDS-Gels.
Preparing crude extract of yeast cells in presence of TCA
To be used for Hansenula polymorpha, Pichia pastoris and Saccharomyces cerevisiae
Published in: Baerends, R.J.S., Faber, K.N., Kram, A.M., Kiel, J.A.K.W., van der Klei, I.J., and Veenhuis, M. (2000) J. Biol. Chem. 275, 9986-9995
Goal: Preparation of protein sample for SDS-PAGE analysis. Proteins are immediately denatured by TCA which prevents enzymatic modification of proteins (including proteolytic degradation, dephosphorylation, deubiquitination, deglycosylation).
Materials: 1. Eppendorf centrifuge
2. -80 oC freezer
3. Vitratron spectrophotometer
Solutions: 1. demineralized water
2. 50 % (w/v) Tri chloro acetic acid
3. cold 80 % (v/v) acetone (cooled at - 20 oC)
4. 1% (w/v) SDS in 0.1 N NaOH
5. 2x SDS sample buffer
Method: 1. Cultivate yeast cells and harvest 3 OD660 units a), spin-down the cells (small volumes up to 2 ml: 30 sec. 14000 rpm, room temp. Eppendorf centrifuge; large samples should be centrifuged in cooled Sorvall centrifuge, 5 min. 5000 rpm, 4o C); remove carefully all supernatant with a pipet. Wash cells once in water (resuspend cells and centrifuge again as above)
2. Resuspend the cells in 300 l water, add 100 μl 50% TCA and mix (alternatively, resuspend cells immediately in 400 μl 12,5 % TCA).
3. Freeze samples at -80 oC for at least 30 min b).
4. Spin down the cells at rT, 14000 rpm (Eppendorf microcentrifute), 5 min.
5. Wash cell pellet twice with 500 l cold 80 % acetone c), spin for 5 min., 14000 rpm, rT.
6. Remove carefully the supernatant and air dry the pellets.
7. Dissolve the pellet in 100 L 1% SDS / 0.1 N NaOH d).
8. Add 100 L 2x SDS sample buffer and boil the samples 5 min. at 100 oC. The protein concentration is approximately 1.5 mg/ml.
Notes: a) 3.0 OD units yield approx. 300 g protein.
b) The samples can be stored at -80 oC for usage later.
c) 1st time resuspend pellet with pipet and 2nd time mix with vortex.
d) At this stage the cell walls dissolve due to the NaOH.
Crude extract by French press
Reference: Florian Salomons
Goal: Disruption of yeast cells by large pressure.
Materials: 1. French press, microbial physiology a)
2. Big (up to 30 ml) or small cell (1-3 ml)b)
3. gloves
4. tubes
5. Box with ice
Solutions: 1. 50 mM potassium phosphate buffer pH 7.2
2. 0.5 M PMSF in DMSO (500x)
3. 2.5 mg/ml leupeptin in water (1000x)
Method: 1. Clean the French Press cell with demi-water and cool it on ice.
2. Sample preparation; harvest the cells by centrifugation (small samples can be centrifuged in eppendorf centrifuge for 1 min. at room temperature. For larger volumes use cooled Sorvall rotor/centrifuge. Spin for 5 min., 5000 rpm at 4o C). Wash the cells once in solution 1 (i.e. resuspend the cells and centrifuge again). Resuspend pellet in approx. 2 x the volume of the cell pellet. Add PMSF (final concentration 1 mM) and leupeptin (final conc 2.5 g/ml).
3. Dry the cell, put the piston in the cell. Than poor the sample in the cell.
4. Adjust the piston till the sample level reaches the tap outlet.
5. Open the tap and put the lid on the cell. Close the tap.
6. Transfer the cell into the French press.
6. Switch the pressure manifold to MEDIUM for the small cell, and HIGH for the big
cell.
7. Increase the pressure with the pressure control. The pressure is NOT allowed to exceed the 1000 PSI for both cells !!!
8. Put an ice-cold tube under the tap and open the tap carefully, so that the cell extract pours out in droplets d) .
9. When this is finished, reduce the pressure to 0, turn the pressure manifold to DOWN and increase the pressure again. When the piston is free from the French press, take out the cell..
10. Repeat step 4. – 10. (2 times is sufficient in most cases). Check by light microscopy whether the cells are broken.
11. Start at 1 for a new sample.
12. To obtain a cell free extract (i.e. only soluble proteins), the samples are centrifuged, for 15 min., 20.000 x g, 4o C.
Notes: a) Also at the French press a user manual is present.
b) Very expensive.
c) Wear gloves since PMSF is toxic.
d) Be careful! When the tap is opened to briskly, the cell suspension flows out to fast
without cell disruption.
The alkaline lysis mentioned by Christian works well in hand, too. It also offers the advantage of being relatively quick and to allow the processing of a subsequent number of samples.
This protocol is used in my lab:
-Spin down cells at 3000rpm for 5 min
-Resuspend in 200ul H2O and mix with 200ul 0.2M NaOH. Incubate at room temp for 5 min (amount can be increased or decreased depending on your pellet size but volumes should be 1:1).
-Centrifuge at max speed (~13,300rpm) for 3 min at room temp and aspirate supernatant.
-Add 50 to 200ul (judging by the size of your pellet) of pre-boiled 1xSDS loading buffer. Boil for 5 min.
-Centrifuge at max speed for 3 min. You can now apply the samples to a gel or store at minus 20C for later use.
This protocol has been used in my lab for quickly checking proteins and always works.
All the suggestions above should work well in your application. One more option for you:-) There is a magic lysis solution you might want to try although it is original designed for RCR or RT-PCR. Please see the details below. It will be on the market very soon! In generally, all you need to do is adding the lysis buffer in the cell pellet (from
1.use Lyticase or Zymolase to make spheroplast.
2. use DEAE-dextran to break plasma membrane but keep intracellular organelles intact.
If you do not care about intracellular organelles, use TX100 to dissolve all membranes.
3. treat solution as total cell extract. Use TCA to precipitate proteins if necessary.
Hello,
I think you can do it easily by two methods:
1) Sonication
2) French press
Normally by sonication, some of the membrane bound proteins remain in the cell body.
But using French press can release most of the proteins. I prefer this methods.
By the way, you should use appropriate lysis buffer.
I have tried lysing yeast by grinding them under liquid nitrogen (LN2) and by using a detergent lysis method (YeastBuster) at 4 deg C and compared these methods with the traditional glass bead method using a vortex. I avoided methods that would raise the temperature above 4 deg C to minimize alterations to the protein I was trying to study, and avoided harsher methods as I intended to use the same method to isolate the protein for biochemical studies. The glass bead method gave me the greatest degree of cell lysis. I noticed that the YeastBuster preps gave me the lowest degree of recovery of nuclear protein, so the nuclear membrane seemed to resist the detergent treatment. Grinding under LN2 proved to be as effective as using glass beads but is more labor intensive and requires some creativity when it comes to small amounts of cells.
Tatiana, if you would like to order some glass beads, those that meet these specifications will work for you: Sigma G-8772
Treat your cells with 2M LiAc (pre-chilled) for 5 minutes on ice. Pellet cells and replace with (pre-chilled) 0,4M NaOH. Incubate for 5 minutes on ice. Pellet the cells and remove NaOH. Resuspend in 2xSDS-PAGE sample buffer and sonicate (required to extract DNA bound proteins). Then boil the sample for 5 minutes and spin down the debris. Transfer the supernatant to a new tube.
Published protocol can be found here
http://onlinelibrary.wiley.com/doi/10.1002/yea.1905/pdf
I added the sonication step.
I found that described method yields considerably more protein than zymolyase incubation followed by boiling in SDS sample buffer.
I hope this helps.
Hi,
use the freeze cracking technique. Take your cells put it in liquid nitrogen for 2 mins followed by an incubation at 37C for 2 mins. Repeat this for 3 times. You will have your protein for WB.
Goodluck!
I want to know initial volume for primary and secondary inoculation.How much cell pellet is required ,volume of LiAC , NaOH ,SDS sample buffer and why there is no need of using protease inhibitor by this method .Plzzz suggest
Preparing crude extract of yeast cells in presence of TCA
To be used for Hansenula polymorpha, Pichia pastoris and Saccharomyces cerevisiae
Published in: Baerends, R.J.S., Faber, K.N., Kram, A.M., Kiel, J.A.K.W., van der Klei, I.J., and Veenhuis, M. (2000) J. Biol. Chem. 275, 9986-9995
Goal: Preparation of protein sample for SDS-PAGE analysis. Proteins are immediately denatured by TCA which prevents enzymatic modification of proteins (including proteolytic degradation, dephosphorylation, deubiquitination, deglycosylation).
Materials: 1. Eppendorf centrifuge
2. -80 oC freezer
3. Spectrophotometer
Solutions: 1. demineralized water
2. 12.5 % (w/v) Tri chloro acetic acid
3. cold 80 % (v/v) acetone (cooled at - 20 oC)
4. 1% (w/v) SDS in 0.1 M NaOH
5. 4x SDS sample buffer
Method: 1. Cultivate yeast cells and harvest 3 OD660 units a), spin-down the cells in 2 ml Eppendorf tubes 1 min 14000 rpm, room temp.
2. Wash cells once in 900 μl water. Vortex cells, centrifuge again as above and remove supernatant.
3. Resuspend the cells in in 400 μl 12,5 % TCA.
3. Freeze samples at -80 oC for at least 30 min b).
4. Spin down the cells at rT, 14000 rpm (Eppendorf microcentrifute), 5 min.
5. Wash cell pellet twice with 500 ml cold 80 % acetone c), spin for 5 min., 14000 rpm, rT.
6. Remove carefully the supernatant and dry the pellets.
7. Dissolve the pellet in 75 mL 1% SDS / 0.1 M NaOH d).
8. Add 25 mL 4x SDS sample buffer and boil the samples 5 min. at 100 oC.
Notes: a) 3.0 OD units yield approx. 300 mg protein.
b) The samples can be stored at -80 oC for usage later.
c) 1st time resuspend pellet with pipet and 2nd time mix with vortex.
d) At this stage the cell walls dissolve due to the NaOH.
For physical grinding, use a bead dispenser to load your vials and plates with your desired amount of beads.
Here is a bead dispenser that I work with. Also using a large number of samples. LabTIE also has a large variety of Bead dispensers, fitting tubes, vials, 96-well 384-well etc.
https://www.labtie.com/seed-bead-powder-dispensers/112346-bead-dispensers
https://www.labtie.com/seed-bead-powder-dispensers/112346-bead-dispensers
Transfer yeast single colony using a sterile pipette tip into a micro-centrifuge tube contianing 500µL dH20. Mixed thoroughly using a vortex for 5 minutes. Add 500µL of 0.2M NaOH (cell membrane pre-treatment). Mixed thoroughly using a vortex for 5 minutes. Incubate suspension at room temperature (~21 degrees Celsius) for 5 minutes. Harvest treated cell by centrifigation (14 000 rpm for 3 minutes). Discard supernatant and add 500µL Urea Lysis Buffer (9M urea, 2M thiourea and 4% CHAPS, PMSF) to pellet. Shake vigorously using a vortex-genie for 1 hour. Store at -20 degrees Celsius until required.
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