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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

STATUS: COMPLETE

Description:

Making pEXPRs hEF1a:LilrB2 and hEF1a:PirB.

 

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
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

Experiment

Protocols:

Immunostaining for flow cytometry

Immunostaining for microscopy

Antibody
Type
Conjugation
Dilution Used
Link
Anti-LilrB2Mouse monoclonal 

Cytometry: 1/2000

Immunofluorescence:

http://www.abcam.com/lilrb2-antibody-ab172538.html
Anti-PirBGoat polyclonal 

Cytometry: 1/2000

Immunofluorescence:

http://www.scbt.com/datasheet-9609-Pirb-c-19-antibody.html

Donkey Anti-Goat IgG

 Alexa Fluor 488

Cytometry: 1/2000

Immunofluorescence: 1/200 - 1/1000.

 
Donkey Anti-Mouse IgG 

Alexa Fluor 488

Cytometry: 1/2000

Immunofluorescence: 1/200 - 1/1000.

 

 

LilrB2

TRIAL 1

Well 1

B Cells

Well 2

HEK 293

 

 

 

Permeabilized

(Unstained)

Well 3

 HEK293


 

 

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

(Unstained)

Well 9

 HEK293

 

 

 

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

(Unstained)

Well 15

 HEK293

 

 

 

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

(Unstained)

Well 21

 HEK293

 

 

 

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

 

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

 

 

Well 10

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Well 12

 


Results:

Microscopy

PermeabilizedNon-permeabilizedNon-permeabilized stained control 
 


Cytometry

Tube 2Tube 3Tube 4Tube 5
  
Tube 8Tube 9Tube 10Tube 11

Overlay of 4 and 5:

hef1a:mKate + dummy hef1a:mKate + hef1a:LilrB2  
+=

 

 

PirB

TRIAL 1

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

 


Results:

Microscopy

ControlPermeabilizedNon-permeabilizedLilrB2 Paper

Cytometry

SSC-A vs FSC-AFSC-H vs FSC-WSSC-H vs SSC-WYellow vs Red
Tube 1Tube 2Tube 3Tube 4
Tube 5Tube 6Tube 7Tube 8

TRIAL 2 

Well 1

B Cells

Well 2

HEK 293

 

 

 

Permeabilized

(Unstained)

Well 3

 HEK293

 

 

 

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

(Unstained)

Well 9

 HEK293

 

 

 

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

(Unstained)

Well 15

 HEK293

 

 

 

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

(Unstained)

Well 21

 HEK293

 

 

 

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

 

 

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

 

 

Well 10

 HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 12

 


 

 



 

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