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.
Controls:
Add antibodies to cells that aren't expressing receptor
Experiments Included:
Experiment A1
Description:
Make HEK293 expressing LilrB2 and PirB on the cell membrane. Will test membrane localization by using anti-LilrB2 and anti-PirB antibodies in Experiment A2. cells- Verify miniprep product
- Transfect into HEK293
Status:
- First and second set of liquid cultures didn't grow, possibly because:
- Too much media
- Plates w
- First and second set of liquid cultures didn't grow, possibly because:
- First set of plates didn't have any blue colonies
- Second set of plates grew blue and white colonies
- PirB didn't grow any white colonies
- Colonies picked for LilrB2
- Liquid culture grew
- Miniprepped - Product to be verified
- Golden Gate repeated
- 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
- LRs with pENTRs made
- 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
- Restriction digest and gel ran to verify LR products
- LR with new PirB pENTR made
Experiment A2
Description:
Testing membrane localization of receptor proteins using fluorophore conjugated anti-LilrB2 and anti-PirB antibodies.
Plan:
- Transfect HEK293 with hEF1a:PirB/LilrB2
- Check for membrane localization using anti-PirB/LilrB2 primary antibodies and secondary antibodies conjugated to fluorphores
Status:
- Anti-LilrB2 and anti-LilrB2 antibodies are not verified for immunostaining
- because they are not verified, we can get them at a generous discount provided that we let abcam know of our results
- we can get an anti-(_)FP antibody and stain for the receptor fused to a fluorescent protein instead
- pursue both simultaneously?
Results:
LilrB2
| Expand | ||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||||||||||||||||||||||||||||
Microscopy
Cytometry
Overlay of 4 and 5:
|
| Expand | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||
|
| Expand | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||
| ||||||||||||
PirB
| Expand | |||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| |||||||||||||||||||||||||||||||||||||
Microscopy
Cytometry
|
| Expand | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||
Well 4 - mKate bleeds into the yellow channel.. a lot.
|
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
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)