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

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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.

简介

该协议描述如何使用NaOH对全酵母细胞样品进行裂解。 然后裂解的细胞可用于下游应用,例如总蛋白的提取。

材料和试剂

  1. NaOH(Sigma-Aldrich)
  2. β-巯基乙醇(β-ME)(Sigma-Aldrich)
  3. 100%(w/v)三氯乙酸(TCA)(Sigma-Aldrich)
  4. 胃酶抑素A(Sigma-Aldrich,目录号:P5318)
  5. 亮肽素(Sigma-Aldrich,目录号:L2884)
  6. PMSF(Sigma-Aldrich,目录号:P7626)
  7. 丙酮(Sigma-Aldrich)
  8. 蛋白酶抑制剂
  9. DMSO
  10. 甘油
  11. BPB
  12. 乙醇
  13. SDS裂解缓冲液
  14. 7.5ml 1M Tris-HCl(pH6.8)(参见配方)
  15. DTT股票(见食谱)
  16. 5x SDS-PAGE凝胶上样染料(见配方)
  17. 蛋白酶抑制剂(参见配方)

程序

  1. 颗粒2 OD的细胞。 用0.5ml水洗涤1次(细胞沉淀可以在此时在液氮中冷冻)。
  2. 重悬浮颗粒在100μl水(确保丸完全重悬)。
  3. 加入17μl的1.85M NaOH/7.4%(v/v)β-ME并涡旋。 在冰上孵育10分钟。
  4. 加入8μl的100%(w/v)TCA,涡旋,并在冰上孵育10分钟。
  5. 在4℃以20,000×g/14,000rpm离心10分钟。 用500μl冷丙酮洗涤蛋白质沉淀(不要上下移动;添加溶液和涡旋;如果沉淀没有完全重悬,则可以)。
  6. 在4℃下再旋转样品5分钟。 几乎完全干燥。 如果颗粒太干,以后不会溶解。
  7. 重悬在100μl的SDS裂解缓冲液+ 1×蛋白酶抑制剂(吸管向上和向下,然后涡旋,然后完全悬浮颗粒)。
  8. 如果进行BCA测定,煮沸5分钟并进行测定
  9. 加入25微升5×SDS-PAGE凝胶加载染料到100微升的样品。
  10. 煮沸5分钟,每个泳道加载40μg总蛋白

食谱

  1. 为了制备1ml NaOH /β-ME,混合741μl水,185μl10N NaOH(储备10N NaOH溶液)和74μlβ-ME原液。 始终制备新鲜的NaOH/beta-ME。
  2. 5x SDS-PAGE凝胶上样染料 150mM Tris-HCl
    15%SDS
    25%甘油 0.02%BPB
    12.5%β-ME
  3. 制备7.5ml 1M Tris-HCl(pH 6.8)
    7.5 g     SDS
    25 ml  50%甘油 10 ml  H sub 2 O
    20 mg  BPB
    最终体积为43.75 ml。 等分到1毫升,加入125微升的β-ME新鲜之前使用。 SDS可能会溶解,但加热会解决这个问题。
  4. 蛋白酶抑制剂
    亮肽素/胃蛋白酶抑制剂A(1mg/ml胃酶抑素A溶于DMSO; 10mg/ml亮抑酶肽溶解于ddH 2 O中)
    合并和分装1毫升每管和存储在-20°C。
    PMSF:0.5M溶于乙醇。 以1ml等分试样在-20℃下储存。 提供1,000x股票。
  5. DTT库存(1 M)
    溶解在ddH 2 O中,以1ml等分试样在-20℃下储存。 是 1,000x股票。

参考文献

  1. Horvath,A。和Riezman,H。(1994)。 从酿酒酵母中快速蛋白提取。 是 10(10):1305-1310。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tong, Z. (2011). Protocol for Whole Cell Lysis of Yeast. Bio-protocol Bio101: e14. DOI: 10.21769/BioProtoc.14;
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Lois Murray
Dalhousie University
what is the recipe for 1XSDS lysis buffer, in step 6 of your procedure?
6/21/2013 9:31:30 AM Reply
Lois Murray
Dalhousie University

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

6/21/2013 9:32:30 AM


Sandra Malmgren Hill
CMB
What is the initial cell volume for this protocol, and is upscaling applicable for increased volume of cell culture?
5/7/2013 5:46:33 AM Reply
Lili Qian
Shanghai Institute of Materia Medica
Why do you use NaOH to lyse cells and what's the function of Beta-mercaptoethanol?
1/7/2013 6:12:26 PM Reply
Zongtian Tong
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

1/10/2013 10:53:48 PM


What is the composition of the SDS lysis buffer (step 6). Please specify
7/4/2012 2:05:55 PM Reply
Zongtian Tong
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

7/4/2012 11:12:26 PM