Description:
In this experiment, we are aiming to test whether or not our receptor proteins localize to the cell membrane. To do that, we will have HEK293 cells express LilrB2 and PirB. We will test for membrane localization of the receptors through immunofluorescence. We will use primary antibodies that will bind to LilrB2 and and PirB and secondary antibodies conjugated to fluorophores that will bind to the primary antibodies.
LilrB2
Results:
Microscopy
| Permeabilized | Non-permeabilized | Non-permeabilized stained control | |
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Cytometry
| Tube 2 | Tube 3 | Tube 4 | Tube 5 |
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| Tube 8 | Tube 9 | Tube 10 | Tube 11 |
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Overlay of 4 and 5:
| hef1a:mKate + dummy | hef1a:mKate + hef1a:LilrB2 | |||
| + | | = |  |
PirB
Results:
Microscopy
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| Control | Permeabilized | Non-permeabilized | LilrB2 Paper |
Cytometry
| SSC-A vs FSC-A | FSC-H vs FSC-W | SSC-H vs SSC-W | Yellow vs Red |
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| Tube 1 | Tube 2 | Tube 3 | Tube 4 |
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| Tube 5 | Tube 6 | Tube 7 | Tube 8 |
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| Negative Control | PirB-YFP only (no transfection marker) |
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Analysis:
Cytometry..
LilrB2 and PirB Trial 1: The plots for the mkate only and mkate + receptor cell populations are very similar. This is most probably due to the bleed through of the red channel into the yellow channel (the 'sensitivity' of the PMTs for the yellow channel was increased because the yellow fluorophore was not very bright).
Next steps:
- We are going to transfect the receptors without using a transfection marker (Trial 2).
Microscopy..
Immunostaining
LilrB2 and PirB Trial 1: For both the receptors, there doesn't seem to be any clear localization of the receptors to the membrane. From the punctate appearance of the cells under the confocal, they seem to be significant localization of the receptors in some subcellular cytoplasmic structure. They may be making it to the membrane; however we are unable to discern that just from the images. Interestingly, this cytoplasmic localization seems to be apparent in the non-permeabilized cells which may mean that the fixing protocol is causing some kind of permeabilization in the cells. It may be of significance to note that in the LilrB2 paper (the one we are trying to replicate), they expressed the receptors under CMV (not hEF1a). Depending on the relative strengths of the promoters, the differing levels of expression (probably higher expression through hEF1a) might be causing the staining pattern that we are seeing.
YFP fusion
maybe the yfp is messing up the structure of the pirb making it not localize to the membrane. idk, it looks like it is not getting expressed at all (no yellow = sadness).
hmmm.... (i should probably write something here)
Next steps:
- Perhaps staining live cells (to skip the fixation step) and hopefully conclude whether or not some of the receptor is making it to the membrane. (We are also going to do the beta amyloid binding experiment despite being unsure whether or not the receptor is making it to the membrane in hopes that it does bind and we can conclude that it is localizing.)
- Once we get the receptor-fluorescent protein fusions out of cloning, we can express them and look for localization. This will tell us whether or not the staining pattern that we're seeing is due to the protein expression or the staining.
- Clone the receptors into a different promoter: CMV or a TRE to determine if high expression levels are the problem. Having inducible expression of the receptors may be helpful later on in determining what the optimal level of receptor expression for the system.