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Immunofluorescence Analysis of Yeast Protein
酵母蛋白的免疫荧光分析   

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Abstract

Many important regulatory proteins such as transcription factors are regulated through subcellular localization. Protein localization can be examined by fusing a GFP tag. However, GFP is relatively big in size, and potentially may affect correct protein localization. Several small tags have been developed, such as myc, HA or Flag. By using immunostain and fluorescence microscopy as described in this protocol, one can easily probe the regulation of a selected yeast protein with the application of the aforementioned small tags.

Keywords: Yeast(酵母), Immunofluorescence(免疫荧光), Microscope(显微镜), Spheroplast(原生质体), Antibody(抗体)

Materials and Reagents

  1. Yeast cells
  2. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P8416 )
  3. Potassium phosphate dibasic (K2HPO4·3H2O) (Sigma-Aldrich, catalog number: P9666 )
  4. Sorbitol (C6H14O6) (Sigma-Aldrich, catalog number: S1876 )
  5. BSA (Albumin from bovine serum) (Sigma-Aldrich, catalog number: A4503 )
  6. Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: BP366-1 )
  7. 37% formaldehyde solution (Thermo Fisher Scientific, catalog number: F75P1GAL )
  8. Zymolyase (USB, catalog number: Z1001 )
  9. Vectorshield
  10. Poly-L-lysine
  11. DAPI
  12. Cytoseal 60
  13. Phosphate buffer (see Recipes)
  14. Sorbitol buffer (see Recipes)
  15. Blocking buffer (see Recipes)

Equipment

  1. Centrifuges
  2. Shaker
  3. Conical tube
  4. Fluorescence microscope
  5. 15 ml conical tube
  6. Light microscope
  7. Heat blot

Procedure

  1. Inoculate yeast cells overnight in a 30 °C shaker.
  2. Subculture cells at OD600=0.1 in 5 ml YPD or defined media.
  3. Continue to shake at 30 °C for 4-6 h to a concentration of 1-5 x 107.
  4. Add 0.6 ml of 37% formaldehyde solution directly to the cells and continue to incubate with shaking for 90 min (to be exact) at the same temperature as growth.
  5. Transfer cells to a 15 ml conical tube and pellet by centrifugation at 660 x g for 3 min at 4 °C.
  6. Aspirate supernatant and wash cells with ice-cold 5 ml phosphate buffer. Be gentle at this step because yeast cells are very fragile after fixation by formaldehyde.
  7. Repellet cells and wash with ice-cold 5 ml sorbitol buffer.
  8. Pellet cells and aspirate supernatant. Resuspend in ~1 ml sorbitol buffer.
  9. Pre-warm the cells at 30 °C for 5 min.
  10. Add 25 μl of zymolyase, mix gently and incubate on 30 °C heat blot for 15-30 min.
  11. Check the digestion under a light microscope (fully digested cells are gray while undigested cells are bright).
  12. When 80% of cells are digested, Pellet cells 660 x g for 3 min at 4 °C. Re-suspend cells gently in 0.5 ml ice-cold sorbitol buffer.
  13. Repeat step 12.
  14. Prepare slides by coating them with Poly-L-lysine for 10 min at room temperature (RT).
  15. Wash slides 5 times with water and let them dry at RT.
  16. Place 20 μl of cell suspension into each well on the slide. Incubate in a wet chamber for at least 10 min.
  17. Immediately immerse slide in ice-cold methanol for 6-7 min.
  18. Remove and immerse immediately in ice-cold acetone for 30 sec.
  19. Dry the slides on 30 °C heat blot.
  20. From this step on, don’t let the wells dry. Wash wells several times with blocking buffer and incubate a moist chamber for at least 10 min.
    Note: longer incubation time may give better results, for example, incubate in blocking buffer at 4 °C overnight.
  21. Remove supernatant and add 20 μl of primary antibody (diluted in blocking buffer). Incubate in a moist chamber for at least 2 h. Note: can put the slides at 4 °C overnight.
  22. Aspirate excess solution and wash 5x with blocking buffer.
  23. Add 20 µl of fluorescence conjugated-secondary antibody (diluted in blocking buffer).
  24. Place in a dark/moist chamber for ~1 h. Remember to keep slides in the dark as much as possible to prevent bleaching of fluorescence.
  25. Aspirate excess solution and wash 5x with blocking buffer.
  26. Then, wash once with 1x PBS.
  27. Dilute DAPI (1:1,000) in 1x PBS.
  28. Add DAPI solution to wells for 2 min.
  29. Aspirate excess solution and wash once with 1x PBS. Do not allow the slide to dry and add 2 µl Vectorshield to the well and immediately cover with cover slide.
  30. Seal the edge of the slides with Cytoseal 60 and examine under a fluorescence microscope.

Recipes

  1. Phosphate buffer
    Make 0.1 M KH2PO4 in H2O
    Make 0.1 M K2HPO4 in H2O
    Add K2HPO4 solution to KH2PO4 solution to bring the pH to 6.5.
  2. Sorbitol buffer
    0.1 M potassium phosphate buffer (pH 6.5)
    1.2 M sorbitol
  3. Blocking buffer
    5 % BSA in 1x PBS (pH 8.0)

Acknowledgments

This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).

References

  1. Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
  2. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.

简介

许多重要的调节蛋白如转录因子通过亚细胞定位进行调节。 蛋白质定位可以通过融合GFP标签来检查。 然而,GFP在尺寸上相对较大,并且潜在地可能影响正确的蛋白质定位。 已经开发了几个小标签,例如myc,HA或Flag。 通过使用如本方案中所述的免疫染色和荧光显微镜,可以通过应用上述小标签容易地探测所选择的酵母蛋白的调节。

关键字:酵母, 免疫荧光, 显微镜, 原生质体, 抗体

材料和试剂

  1. 酵母细胞
  2. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P8416)
  3. 磷酸氢二钾(K 2 HPO 4·3H 2 O)(Sigma-Aldrich,目录号:P9666)
  4. 山梨醇(C 6 H 14 O 6)(Sigma-Aldrich,目录号:S1876)
  5. BSA(来自牛血清的白蛋白)(Sigma-Aldrich,目录号:A4503)
  6. 氯化钾(KCl)(Thermo Fisher Scientific,目录号:BP366-1)
  7. 37%甲醛溶液(Thermo Fisher Scientific,目录号:F75P1GAL)
  8. 酵母聚糖酶(USB,目录号:Z1001)
  9. 矢量屏蔽
  10. 聚-L-赖氨酸
  11. DAPI
  12. Cytoseal 60
  13. 磷酸盐缓冲液(参见配方)
  14. 山梨醇缓冲液(见配方)
  15. 阻止缓冲区(参见配方)

设备

  1. 离心机
  2. 振动器
  3. 圆锥管
  4. 荧光显微镜
  5. 15 ml锥形管
  6. 光学显微镜
  7. 热印

程序

  1. 在30℃振荡器中接种酵母细胞过夜
  2. 在5ml YPD或确定培养基中OD 600 = 0.1时的亚培养细胞
  3. 在30℃下继续振摇4-6小时至1-5×10 7的浓度
  4. 将0.6ml的37%甲醛溶液直接加入到细胞中,并在与生长相同的温度下继续振荡孵育90分钟(准确地)。
  5. 将细胞转移到15ml锥形管中,并通过在4℃下以660×g离心3分钟沉淀。
  6. 吸出上清液,用冰冷的5 ml磷酸盐缓冲液洗涤细胞。 在这一步温和,因为酵母细胞在甲醛固定后非常脆弱
  7. Repellet细胞并用冰冷的5ml山梨醇缓冲液洗涤
  8. 沉淀细胞并吸出上清液。 重悬于〜1ml山梨醇缓冲液中
  9. 在30℃预热细胞5分钟。
  10. 加入25μl酶解酶,轻轻混合,并在30℃热印迹孵育15-30分钟
  11. 在光学显微镜下检查消化(完全消化的细胞是灰色而未消化的细胞是明亮的)
  12. 当80%的细胞被消化时,沉淀细胞在4℃下660×g离心3分钟。 在0.5ml冰冷的山梨醇缓冲液中轻轻重悬细胞
  13. 重复步骤12.
  14. 通过在室温(RT)下用聚-L-赖氨酸涂覆载玻片10分钟来制备载玻片
  15. 用水冲洗载玻片5次,并在室温下使其干燥
  16. 放置20微升的细胞悬浮液在载玻片上的每个孔。 在湿室中孵育至少10分钟。
  17. 立即将载玻片浸入冰冷的甲醇中6-7分钟。
  18. 取出并立即浸在冰冷的丙酮中30秒。
  19. 在30°C热印迹上干燥载玻片。
  20. 从这一步,不要让井干。 用封闭缓冲液洗涤孔几次,并孵育潮湿室至少10分钟。
    注意:更长的温育时间可以产生更好的结果,例如,在封闭缓冲液中在4℃孵育过夜。
  21. 除去上清液,加入20μl一抗(在封闭缓冲液中稀释)。 在潮湿的室内孵育至少2小时。 注意:可以将幻灯片在4°C过夜。
  22. 吸出过量的溶液,用封闭缓冲液洗5次。
  23. 加入20微升荧光共轭二级抗体(在封闭缓冲液中稀释)。
  24. 置于黑暗/潮湿的室内约1小时。 记住要尽可能保持幻灯片在黑暗中,以防止漂白的荧光。
  25. 吸出过量的溶液,用封闭缓冲液洗5次。
  26. 然后,用1x PBS洗涤一次。
  27. 在1x PBS中稀释DAPI(1:1,000)。
  28. 将DAPI溶液加入孔中2分钟。
  29. 吸出过量的溶液,并用1x PBS洗涤一次。 不要让幻灯片干燥,并添加2μl的VectorHield到井,立即盖玻片。
  30. 用Cytoseal 60密封载玻片的边缘,并在荧光显微镜下检查

食谱

  1. 磷酸盐缓冲液
    在H 2 O中制备0.1M KH 2 PO 4子
    在H 2 O中制备0.1M K 2 HPO 4子
    将K 2 HPO 4溶液加入KH 2 PO 4溶液中以使pH达到6.5。< br/>
  2. 山梨醇缓冲液
    0.1M磷酸钾缓冲液(pH6.5) 1.2M山梨醇
  3. 阻塞缓冲区
    5%BSA的1x PBS(pH 8.0)中洗涤

致谢

该方案改编自并用于Wei和Zheng(2009)和Wei et al。 (2009)。

参考文献

  1. Wei,Y。和Zheng,X.F。(2009)。 Sch9 部分介导TORC1信号传导以控制核糖体RNA合成。 Cell Cycle 8(24):4085-4090。
  2. Wei,Y.,Tsang,C.K.and Zheng,X.F。(2009)。 TORC1调节RNA聚合酶III依赖性转录的机制。 EMBO J 28(15):2220-2230。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wei, Y. (2012). Immunofluorescence Analysis of Yeast Protein. Bio-protocol 2(13): e211. DOI: 10.21769/BioProtoc.211.
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Shivani Goolab
CSIR
Immunofluorescence imaging Y. lipolytica cells:

I assessed unpermeabilized, saponin permeabilized and triton x-100 permeabilized untransformed cells (as a negative control) and as the test my gene expressed in Y. lipolytica. Using the FITC channel, autofluorescence of the cells is evident in both the negative and test. Is this common for yeast?
12/15/2016 2:15:34 AM Reply
Yuehua Wei
Shanghai Jiao Tong University

Hi Shivani,

From what you described, I think it is likely that the signals are from non-specific binding of antibody. Yes, this non-specific signals are very common in yeast, and also in human cells when doing immuno-fluorescence experiments. So one has to manage very well about the dilution of the antibody, by using the right concentration of both primary and secondary antibody and by washing away the non-specific binding by certain about of detergent.

You did great by adding non-transformed cells as a negative control, which tells me right away that the signal is very likely from non-specific binding of the antibody. You can further figure out if it is the primary antibody or secondary antibody that cause such non-specific binding, by adding another controls that add no primary antibody or add no secondary antibody. If the signal is gone, you then know it is from the which antibody. From that point, you can dilute your antibody 10X, 100X etc. If the signals still go weaker together in transformed and non-transformed cells, it will suggest that likely your washing buffer is not optimal. If this is the case, you can increase detergents in your washing buffer. Sometimes increasing salt concentration in washing buffer also helps.

Hope that my answers will help. Thank you for the question and good luck with you experiment!

YW

12/23/2016 9:29:01 AM


Shivani Goolab
CSIR

Dear Yuehua Wei

Thank you for your guidance. Please find attached image of non-transformed cells before treatment with the primary and secondary Ab. The signal persists even before adding Abs. I thought it may have been media composition since I use YPD media but cells are washed thoroughly in 100mM phosphate wash buffer. You mentioned wash buffer too, I will alter buffer composition to see if any difference is observed.

Once again thank you, I really appreciate the input.

2/12/2017 11:55:52 PM


Yuehua Wei
Shanghai Jiao Tong University

Hi Shivani,
It seems to me that the signals are quite strong, and distributed unequally in mother cells and daughter cells, suggesting that it is not likely to be non-specific binding of antibody. You mentioned that this happens even before adding antibody, confirming that this is not antibody issue. I have not found any of this kind of signal before. Therefore, I guess there must be something different from the common issues I mentioned before. So please don't rush to do antibody or detergent dilution.
I would like to check if the strain itself is bearing any GFP, if I find in my control this "nice" signal. What you can do is to get several strains that you are sure having no GFP, culture them in the same media and examine directly. If only this strain showing green but not others, it will suggest that you have a GFP in your strain. You can also examine under RFP channel, if this signal is gone, likely this is due to GFP in your strain. If all strains have the same signals, then your initial hypothesis are likely right: something in the media causes the signal. Then you will need to go through your media protocol carefully to see if you have some thing different. But a more easy way is to buy a ypd from a company or get them from other labs. Some times it is really hard to find out the contaminants. Thanks for sharing and Let me know if you have further questions.

2/14/2017 1:26:40 PM