Description:
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. We will then apply biotinylated anti-LilrB2 and anti-PirB antibodies to the cells. To test for localization, we will use the biotin-binding protein streptavidin covalently bonded to a fluorophore.
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.
Plan:
- Order LilrB2 and PirB gBlocks
- Simulate Golden Gate on Geneious and make sure it works
- Golden Gate gBlocks into ggDONR
- Transform bacteria with Golden Gate product and plate
- Pick colonies and grow in liquid culture
- Miniprep cells
- Verify miniprep product
- LR into pDEST
- Transfect into HEK293
Status:
- First and second set of liquid cultures didn't grow, possibly because:
- Too much media
- Plates were old, antibiotic possibly not working
- 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
Experiment A2
Description:
Testing membrane localization of receptor proteins using biotinylated anti-LilrB2 and anti-PirB antibodies.
Plan:
Status:
Not yet started. Waiting on Experiment A1.