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.
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Status:
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- First and second set of liquid cultures didn't grow, possibly because:
- Too much media
- Plates were old, antibiotic possibly not working
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- cells transformed and plated
- +ve control didn't grow
- LilrB2 grew blue and white colonies
- PirB grew white colonies
- Both liquid cultures grew
- Miniprepped - Product to be verified
- pENTRs sent for sequencing - waiting on results
- LilrB2 pENTR is correct
- PirB pENTR not correct - Q-Q ligation, pENTR only has C terminus end of PirB
- New PirB pENTR colonies picked, grown and miniprepped
- Restriction digest ran - correct band pattern for one of the colonies
- Correct colony sent out for sequencing - pENTR has correct sequence
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- Restriction digest and gel ran to verify LR products
- LilrB2 gave inconclusive results
- possible incomplete digestion
- PirB results did not match expected band pattern
- Due to incorrect pENTR
- LilrB2 gave inconclusive results
- LilrB2 pEXPR sent out for sequencing
- incomplete sequencing results
- LilrB2 insert is in the pEXPR
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- Restriction digest and gel ran to verify LR products
- PirB insert is in the pEXPR
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LilrB2
TRIAL 1
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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 | |||
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PirB
TRIAL 1
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| Control | Permeabilized | Non-permeabilized | LilrB2 Paper |
Cytometry
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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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TRIAL 2
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| title | Trial 2 |
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| title | Cytometry Plate |
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Results:
LilrB2
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Microscopy
Cytometry
Overlay of 4 and 5:
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PirB
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Microscopy
Cytometry
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Well 4 - mKate bleeds into the yellow channel.. a lot.
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Analysis:
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Well 1
B Cells
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Well 2
HEK 293
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Well 3
HEK293
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Well 4
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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Well 5
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:PirB (500ng)
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Well 6
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Well 7
B Cells
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Well 8
HEK 293
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Well 9
HEK293
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Well 10
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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Well 11
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:PirB (500ng)
Well 12
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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.
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.
YFP fusion
Evidence is not super convincing that the yellow that we are seeing is not just autofluorescence given that we see something very similar in our negative control. It is likely that PirB is not getting expressed at all, or that the fusion is disturbing it's structure somehow making it not localize to the membrane (the happier scenario). mKate bleedthrough into the yellow channel was an issue. Ideally, we would make an eBFP fusion but considering how hard it was to make the fusions in the first place, that isn't really a feasible solution, we can possibly look into using an IR protein as a transfection marker (Trial 4). Also, it is probably a good idea to DAPI counterstain next time. Zstacks with nuclear staining will help in determining membrane localization (or lack thereof).
Next steps:
- Is the receptor even getting expressed? Maybe if we transfect TRE:receptor-YFP and run a dox ladder, we can conclude on whether or not there is expression based on whether or not there are varying levels of yellow corresponding to the dox ladder.
- Run PirB Trial 3 again but with an IR fluorescent transfection marker to try and eliminate bleedthrough (Trial 4)