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Immunofluorescence Analysis of Yeast Protein   

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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, Immunofluorescence, Microscope, Spheroplast, Antibody

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.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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
No.3 People's Hospital, Shanghai Jiao Tong University, China

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
No.3 People's Hospital, Shanghai Jiao Tong University, China

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