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In this group of experiments, 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. To We will test for localization, we will use fluorophore conjugated anti-LilrB2 and anti-PirB antibodies. 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
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- 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 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
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