We are trying to introduce new technique in our lab - to see localisation of transcription factors in yeast cell, by tagging these genes and visualizing them by help of secondary antibodies. So far all the "positive controls" f. ex. Msn4 and rapamycine treatment have resulted with granules in cytoplasm as opposed to signal in nucleus. See attached picture - nucleus - DAPI stained, blue, Msn4 C terminally His Tagged, Alexa Red anti mouse secondary antibody
We were wondering, what are we doing wrong?
Thank you in advance
Hi,
we chose to do indirect assessment of localisation because we were afraid that copy number of transcription factor would be too low to detect GFP signal and GFP itself could interfere with localisation. As the method per se (IF for transcr. factors) is published by Cooper group, we think that we are doing something wrong during sample preparation.
But if problems will continue, we will look in to co-localisation. It is a good idea how to check it. Thanks!
Hi Agnese,
The first thing you have to do is including "untagged" control, which will tell you whether the cytoplasmic signal is "background noise" antibodies or indeed from the tagged protein. Depending on the outcome, you can optimise the antibody concentration either by diluting more, or less (or changing the blocking condition).
Immunofluorescence staining in yeast cell is still a "black magic" mainly because of the cell wall. It is quite difficult to obtain convincing staining reproducibly. Particularly if the localisation of your protein of interest is not fully understood, and the expression level of the protein is low, it is really hard to obtain convincing data.
Therefore, if possible, GFP-fusion in live cells is generally more reliable than immunofluorescence.
- Badges
- Science topic