Group A: Membrane Localization

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 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 

Cloning:

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
• Transform, pick colonies and grow in liquid culture
• Miniprep product
• Midiprep product
• Transfect into HEK293

Status:

• • First and second set of liquid cultures didn't grow, possibly because:
    • Too much media
    • Plates w
    ere 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
  • 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 pEXPR sent out for sequencing 
    •  incomplete sequencing results 
    • LilrB2 insert is in the pEXPR
• LR with new PirB pENTR made 

Experiment

Description:

Testing membrane localization of receptor proteins using anti-LilrB2 and anti-PirB primary antibodies and Alexa-Fluor conjugated secondary antibodies. 

In the immunostaining protocol, we are planning to permeabilize our HEK cells 

Transfection Plan:

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?