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
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).
References
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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
Dear Yuehua WeiThank 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.
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.