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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.

 

Expand
titleProgress = COMPLETE
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

 

Expand
titleAntibodies
Antibody
Type
Conjugation
Dilution Used
Link
Anti-LilrB2Mouse monoclonal 

Cytometry:

Immunofluorescence:

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

Cytometry:

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

Expand
titleTrial 1 Transfection Plan
Expand
titleMicroscopy Plate
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
Expand
titleCytometry Plate
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:

Expand
titleLilrB2 Trial 1

Microscopy

PermeabilizedNon-permeabilizedNon-permeabilized stained control 
Image ModifiedImage ModifiedImage Modified 


Cytometry

Tube 2Tube 3Tube 4Tube 5
Image ModifiedImage ModifiedImage ModifiedImage Modified
  Image ModifiedImage Modified
Image ModifiedImage ModifiedImage ModifiedImage Modified
Tube 8Tube 9Tube 10Tube 11
Image ModifiedImage ModifiedImage ModifiedImage Modified

Overlay of 4 and 5:

hef1a:mKate + dummy hef1a:mKate + hef1a:LilrB2  
Image Modified+Image Modified=Image Modified

 

 

PirB

TRIAL 1

Expand
titleTrial 1 Transfection Plan
Expand
titleMicroscopy Plate
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
Expand
titleCytometry Plate
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:

Expand
titlePirB Trial 1

Microscopy

Image AddedImage AddedImage AddedImage Added
ControlPermeabilizedNon-permeabilizedLilrB2 Paper

Cytometry

Expand
titleGating
SSC-A vs FSC-AFSC-H vs FSC-WSSC-H vs SSC-WYellow vs Red
Image AddedImage AddedImage AddedImage Added
Tube 1Tube 2Tube 3Tube 4
Image AddedImage AddedImage AddedImage Added
Tube 5Tube 6Tube 7Tube 8
Image AddedImage AddedImage AddedImage Added

 

TRIAL 2 

Expand
titleTrial 2
Expand
titleMicroscopy Plate
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
Expand
titleCytometry Plate
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

...

 

...

Results:

...

titlePirB Trial 1

Microscopy

Image RemovedImage RemovedImage RemovedImage Removed
ControlPermeabilizedNon-permeabilizedLilrB2 Paper

Cytometry

Expand
titleGating
SSC-A vs FSC-AFSC-H vs FSC-WSSC-H vs SSC-WYellow vs Red
Image RemovedImage RemovedImage RemovedImage Removed

...