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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. 

Cloning

COMPLETE

Description:

Making pEXPRs hEF1a:LilrB2 and hEF1a:PirB.

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 and verify product 
  • Make glycerol stocks
  • LR into pDEST
  • Transform, pick colonies and grow in liquid culture
  • Miniprep cells and verify product
  • Make glycerol stocks 
  • Midiprep product

 

 

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
    • 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 
    • Restriction digest and gel ran to verify LR products 
      • PirB insert is in the pEXPR

 

 

 

 

Experiment

Description:

Testing membrane localization of receptor proteins using anti-LilrB2 and anti-PirB primary antibodies and secondary antibodies conjugated to yellow Alex Fluors.

Transfection Plan:

LilrB2

Well 1

B Cells

Well 2

HEK 293

 

 

 

Permeabilized

Well 3

 HEK293

+ Dummy

(1000ng)

 

Permeabilized

Well 4

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Permeabilized

Well 6
Well 7

B Cells

Well 8

HEK 293

 

 

 

Permeabilized

Well 9

 HEK293

+ Dummy

(1000ng)

 

Permeabilized

Well 10

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Permeabilized

Well 12

 

Well 13

B Cells

Well 14

HEK 293

 

 

 

Non-Permeabilized

Well 15

 HEK293

+ Dummy

(1000ng)

 

Non-Permeabilized

Well 16

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non-Permeabilized

Well 17

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Non-Permeabilized

Well 18
Well 19

B Cells

Well 20

HEK 293

 

 

 

Non-Permeabilized

Well 21

 HEK293

+ Dummy

(1000ng)

 

Non-Permeabilized

Well 22

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non-Permeabilized

Well 23

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Non-Permeabilized

Well 24
Well 1

B Cells

Well 2

HEK 293

 

 

 

Well 3

 HEK293

+ Dummy

(1000ng)

Well 4

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Well 6
Well 7

B Cells

Well 8

HEK 293

 

 

Well 9

 HEK293

+ Dummy (1000ng)

 

Well 10

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Well 12

 


 

PirB

Well 1

B Cells

Well 2

HEK 293

 

 

 

Permeabilized

Well 3

 HEK293

+ Dummy

(1000ng)

 

Permeabilized

Well 4

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Permeabilized

Well 6
Well 7

B Cells

Well 8

HEK 293

 

 

 

Permeabilized

Well 9

 HEK293

+ Dummy

(1000ng)

 

Permeabilized

Well 10

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Permeabilized

Well 12
Well 13

B Cells

Well 14

HEK 293

 

 

 

Non-Permeabilized

Well 15

 HEK293

+ Dummy

(1000ng)

 

Non-Permeabilized

Well 16

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non- Permeabilized

Well 17

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Non-Permeabilized

Well 18
Well 19

B Cells

Well 20

HEK 293

 

 

 

Non-Permeabilized

Well 21

 HEK293

+ Dummy

(1000ng)

 

Non-Permeabilized

Well 22

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non- Permeabilized

Well 23

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Non-Permeabilized

Well 24
Well 1

B Cells

Well 2

HEK 293

Well 3

 HEK293

+ Dummy (1000ng)

 

Well 4

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 6
Well 7

B Cells

Well 8

HEK 293

Well 9

 HEK293

+ Dummy (1000ng)

 

Well 10

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 12

 


 

Status: 

  • First trial with PirB done 
  • Received LilrB2 antibodies - seeding for transfection on 30 Jul

 

 

Results:

PirB First Trial:

Microscopy

ControlPermeabilizedNon-permeabilized

Cytometry

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