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This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins.

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[Bio101] Protocol for Whole Cell Lysis of Yeast
[Bio101] 酵母全细胞裂解技术

生物化学 > 蛋白质 > 电泳
作者: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
1/5/2011, 17097 views, 4 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.14

[Abstract] This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins.

Materials and Reagents

  1. NaOH (Sigma-Aldrich)
  2. Beta-mercaptoethanol (beta-ME) (Sigma-Aldrich)
  3. 100% (w/v) trichloroacetic acid (TCA) (Sigma-Aldrich)
  4. Pepstatin A (Sigma-Aldrich, catalog number: P5318)
  5. Leupeptin (Sigma-Aldrich, catalog number: L2884)
  6. PMSF (Sigma-Aldrich, catalog number: P7626)
  7. Acetone (Sigma-Aldrich)
  8. Protease inhibitors
  9. DMSO
  10. Glycerol
  11. BPB
  12. Ethanol
  13. SDS lysis buffer
  14. 7.5 ml 1 M Tris-HCl (pH 6.8) (see Recipes)
  15. DTT stock (see Recipes)
  16. 5x SDS-PAGE gel loading dye (see Recipes)
  17. Protease inhibitors (see Recipes)

Procedure

  1. Pellet 2 OD of cells. Wash 1x with 0.5 ml of water (cell pellets may be frozen in liquid nitrogen at this point).
  2. Resuspend pellets in 100 μl of water (make sure pellets are fully resuspended).
  3. Add 17 μl of 1.85 M *NaOH/7.4% (v/v) beta-ME and vortex. Incubate on ice for 10 min. 
  4. Add 8 μl of 100% (w/v) TCA, vortex, and incubate on ice for 10 min. 
  5. Spin extract for 10 min at 20,000 x g /14,000 rpm at 4 °C. Wash protein pellet with 500 μl of cold acetone (do not pipette up and down; add the solution and vortex; it is OK if the pellet is not completely re-suspended). 
  6. Re-spin sample for 5 min at 4 °C. Dry almost completely. If the pellet gets too dry, it will not dissolve well later.
  7. Resuspend pellet in 100 μl of SDS lysis buffer + 1x protease inhibitors (pipette up and down, then vortex, then resuspend the pellet completely).
  8. If doing a BCA assay, boil for 5 min and perform the assay.
  9. Add 25 μl of 5x SDS-PAGE gel loading dye to 100 μl of sample.
  10. Boil for 5 min and load 40 μg total proteins per lane.

Recipes

  1. To make 1 ml of NaOH/beta-ME, mix 741 μl of water, 185 μl of 10 N NaOH (stock 10 N NaOH solution) and 74 μl of beta-ME stock. Always make fresh NaOH/beta-ME.
  2. 5x SDS-PAGE gel loading dye
    150 mM Tris-HCl
    15% SDS
    25% glycerol
    0.02% BPB
    12.5% beta-ME
  3. To make 7.5 ml 1 M Tris-HCl (pH 6.8)
    7.5 g    SDS
    25 ml   50% glycerol
    10 ml   H2O
    20 mg  BPB
    Bring final volume to 43.75 ml. Aliquot into 1 ml and add 125 μl of beta-ME fresh prior to use. SDS may come out of solution, but heating will fix this.
  4. Protease Inhibitors
    Leupeptin/pepstatin A (1 mg/ml pepstatin A dissolved in DMSO; 10 mg/ml leupeptin dissolved in ddH2O)
    Combine and aliquot 1 ml per tube and store at -20 °C.
    PMSF: 0.5 M dissolved in ethanol. Store in 1 ml aliquots at -20 °C. Supplied as 1,000x stock.
  5. DTT stock (1 M)
    Dissolve in ddH2O, store in 1 ml aliquots at -20 °C. I.e. 1,000x stock.

References

  1. Horvath, A. and Riezman, H. (1994). Rapid protein extraction from Saccharomyces cerevisiae. Yeast 10(10): 1305-1310.


How to cite this protocol: Tong, Z. (2011). Protocol for Whole Cell Lysis of Yeast. Bio-protocol Bio101: e14. DOI: 10.21769/BioProtoc.14; Full Text



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6/21/2013 9:31:30 AM  

Lois Murray
Dalhousie University

what is the recipe for 1XSDS lysis buffer, in step 6 of your procedure?

6/21/2013 9:32:30 AM  

Lois Murray
Dalhousie University

Oops: should have read to the end of the posts: the recipe is there. Thanks

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5/7/2013 5:46:33 AM  

Sandra Malmgren Hill
CMB

What is the initial cell volume for this protocol, and is upscaling applicable for increased volume of cell culture?

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1/7/2013 6:12:26 PM  

Lili Qian
Shanghai Institute of Materia Medica

Why do you use NaOH to lyse cells and what's the function of Beta-mercaptoethanol?

1/10/2013 10:53:48 PM  

Zongtian Tong (Author)
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

To use NaOH to lyse cell wall: a-glucan and some forms of b-glucan of the yeast cell wall are soluble under alkaline conditions.

The function of beta-mercaptoethanol: reducing agents, such as dithiothreitol or b-mercaptoethanol are needed because the breakage of disulphide bridges between mannose residues and wall proteins is necessary for appropriate exposition of the inner glucan layer.

References:

1. Horvath A, Riezman H. 1994. Rapid protein extraction from Saccharomyces cerevisiae. Yeast 10: 1305–1310

2. Zhang T, et al. 2011. An improved method for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast. 28(11):795-8

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7/4/2012 2:05:55 PM  

What is the composition of the SDS lysis buffer (step 6). Please specify

7/4/2012 11:12:26 PM  

Zongtian Tong (Author)
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

SDS lysis buffer recipe: 1% SDS, 10mM EDTA and 50mM Tris, pH 8.1

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