Protein extraction from yeast(酵母蛋白质提取)

Protein extraction from yeast

1. Glass beads lysis

Conzelmann A, Riezman H, Desponds C, Bron

C.

1988. A major 125 kd membraneglycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through aninositol-containg phospholipid. EMBO J 7: 2233±

2240.

2. rapid protein extraction in optimized SDS sample buffer

HorwathA,RiezmanH.

1994.RapidproteinextractionfromSaccharomycescerevisiae. Yeast 10: 1305±

1310.

Sample Buffer:

0.06M Tris-HCl, pH

6.8

10% (v/v) glycerol

2% (w/v) SDS

5% (v/v) 2-mercaptoethanol

0.0025% (w/v) bromophenol

blue10 ml

0.6 ml 1M Tris

6.8

2 ml 50% glycerol

2 ml 10% SDS

0.5 ml 2-mercaptoethanol

0.1 ml Sat. Bromphenol

blue

4.9 ml H2O

Make sample Buffer fresh before use. Can store buffer frozen at—20 degrees for ~6 months.

1. Grow cells overnight (~1x107cells/ml; A600 =

0.7) and collect

1.5 ml cells (adjustvolumes according to cell density of cultures) in

1.5 ml microfuge tube (1 minute, 14000xg).It is important not to grow the cells to a high density.

10 microliters of a saturated overnight culture in YPD innoculated to 5 ml SD + essentialamino acids for ~16 hrs gives A600 of

0.5 to

1.0 for wild-type cells grown at 30 degrees150 microliters of YPD saturated culture diluted to 5 ml YPD and grown for ~5 hrs at 30degrees gives an A600 of ~

0.8 for wild-type cells.

2. Wash cells 1X with water and collect again by centrifugation.

3. Resuspend cells in 100 microliters sample buffer.

4. Heat at 95 deg C for 5 minutes.

5. Centrifuge 14000xg for 5 minutes. Load 15 microliters per lane on an SDS PAG

E3. post-alkaline extraction

VitalyV.Kushnirov

2000.Rapidandreliableproteinextractionfromyeast.Yeast2000; 16: 857±

860.

about

2.5 OD600 (which constitutes about

2.3 mg of wet weight) of yeast cells wereharvested by centrifugation from liquid culture or scraped off the agar plate using

abacteriologicalloop.Thesecellsweresuspendedin100mldistilledwater,added100 ml

0.2 M NaOH, incubated for 5 min at room temperature, pelleted, resuspendedin50mlSDSsamplebuffer,boiledfor3minandpelletedagain.About6μlsuper natantwastypicallyloadedperlaneofmini-gel(Bio-RadMini-

Proteancell).Thesamplebuffer(

0.06MTris±HCl,pH

6.8,5%glycerol,2%SDS,4%bmercaptoethanol,

0.0025% bromophenolblue) was slightly modi?ed fromstandard(Laemmli, 1970).

SDS-Boil-Beads Whole Cell Extracts

REFERENCE:

Hoffman,G.,Garrison,T.R.,andDohlman,H.G.,AnalysisofRGS proteins inSaccharomyces cerevisiae, Methods Enzymol.344:617-631,

2002.

-Use sterile technique and sterile solutions in steps 1 to

3.-

https://www.360docs.net/doc/066963104.html,ingasaturatedstarterculture,inoculate25to30mlofappropriatemedia in a 125 ml flask.

***Since it is often difficult to estimate the growth rate of yeast, itishelpfultostartseveral25mlcultures,eachwithadifferentdilutionof the starter culture (e.g. 1:100, 1:300, 1:900).

2.Growat30Cshaking(250rpm)untiltheOD600nm~

1.0(Thisusuallydone overnight).

***When growingseveral strains atonce, itis likely thatthey will

allreachOD600nm~

1.0atdifferenttimes.Ifdesired,sodiumazide(1Mstockin water, diluted to a final concentration of 10 mM) can be added to acultureonceitreachesanOD600nm~

1.0.Theculturecanthenbeplacedon ice until the others are ready.

3. Transfer to a 50 ml conical tube and centrifuge for 10 min at 2000 xg at 4

C.

4. Resuspend each sample in 1 ml of 10 mM sodium azide and place on ice.

5. Calculate the volume of resuspended cells that would translate to anOD600 nm reading of

10. For example, this would equal 1 ml if 10 ml ofculture at OD600 nm =

1.0 had been centrifuged and resuspended.***This step is necessary to equalize the amount of cells (and protein)in a given volume of whole cell extract.

6.Transferthecalculatedvolumeofresuspendedcellstoamicrofugetubeand centrifuge at 16,000 x g for 1 min.

7. Aspirate the supernatent.

8. Resuspend the pellet in 200 ul of 1X SDS-PAGE sample buffer.

9. Immediately place in a 100 C heat block for 10 min.

10.Allowthetubetocoolandadd200ulofglassbeads(Sigma,#G-8772).

11. Vortex at high speed for 2 min. Invert after the first min.***Several tubes can be vortexed at the same time by using a foam tubefloater to hold them together.

12. Using a 21 gauge needle, poke a hole in the bottom of each tube andplace it into a new microfuge tube.

13.Centrifugeat2000xgfor10sectoexpeltheliquidintothebottomtube, leaving the glass beads in the top tube.

14. Discard the glass beads and centrifuge the bottom tube at 16,000 xg for 2 min. This sediments any insoluble material.

15. Transfer the supernatant to a new microfuge tube. Store at -20

C.

16. When ready to use, heat at 37 C for 10 min, vortex, and centrifugeat 16,000 x g for 1 min.

***Keepinmindthatrepeatedfreezingandthawingcandegradetheproteinsample.

17. Immunoblots can be performed using standard methods.

Small Scale Yeast Whole Cell Extract for IP

Steve HahnAugust 2007

Grow 100 ml yeast cells in desired media overnight to an A600 of ~

1.0 (

0.6 to

1.2 works

well).Forgrowthinminimalmedia,1mlofasaturatedovernightcultureinminimalmedia(Sy

nthetic dextrose (SD) with only the required amino acids) was inoculated to 100 ml of thesame media and grown ~16 hrs at 30 degrees.

Harvest cells and wash with 20 ml of cold extraction buffer in a 50 ml tube.

Resuspendcellsin

0.5mlextractionbuffercontaingDTTandproteaseinhibitorsinamicrocentrifuge tube with a locking top (marsh tube).

Add ~500 microliters of glass beads. In the cold room, shake tubes on the foam ring of thevortex mixer platform at top speed for 1 min. Transfer to ice for 1 min. I have done up to 20extracts at once.

Repeat for a total of 10 min of vortexing.

Briefly microcentrifuge to remove all liquid and leave behind most of the glass beads.Centrifuge at top speed at 4 degrees for 15 min and remove supernatant, being careful toavoid any glass beads.

Assay protein concentration using BioRad or Pierce assays. Freeze extracts and store at -80deg. Expect 10-15 mg/ml protein.

Extract Buffer:

100 mM Tris pH

7.9

250 mM Ammonium Sulfate

1 mM EDTA

10% Glycerol

Before use, add DTT to

0.5 mM (low concentration so IP reactions can be done directly)And 1X protease inhibitors from the following stock solutions:

0.1 M PMSF (100x) 16 mg/ml Ethanol; Store at -20 degrees

Benzamidine (100X); 31 mg/ml H2O; Store frozen at -20 degrees

Leupeptin (500X);

0.15 mg/ml Ethanol; Store at -70 degrees for less than 6 monthsPepstatin (200X);

0.28 mg/ml methanol; Store at -20 degrees.

Chymostatin (2,500X); 5mg/ml DMSO; Store frozen at -20 degrees

TCA protein precipitation

To concentrate proteins for analysis by SDS PAGE:

If a small amount of protein is to be precipitated (less than a few micrograms), add Insulin asa carrier protein (10 micrograms of Sigma insulin, I-5500, per sample works well).

1.Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample.

2.Incubate 30 min on ice.

3.Spin in microfuge at 4 deg. For 15 min.

4.Carefully remove all supernatant.

5.Add ~300 ul cold acetone and spin 5 min at 4 degrees.

6.Remove supernatant and dry pellet.

7.Resuspend samples in SDS PAGE loading buffer. Load to SDS PAGE after heating at65 deg for 3 min.

Acetone precipitation of protein

1. Cool the required volume of acetone to -20°

C.

3. Add six times the sample volume of cold (-20°C) acetone to the tube.

4. Vortex tube and incubate for 2 hours to overnight minutes at -20°

C.

5. Centrifuge 15 minutes at 13,000-15,000 x g at 4°

C.

6. Decant and properly dispose of the supernatant, being careful to not dislodge the proteinpellet.

7. Briefly wash the pellet with 100ul of cold 90% acetone.

8. Centrifuge 5 minutes at 13,000-15,000 x g at 4°

C.

9. Remove sup and repeat if necessary.

10. Air dry for ~15-30 minutes and resuspend in an appropriate buffer.