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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

Expand

title	Progress = 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

Description:

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

Antibody	Type	Conjugation	Dilution Used	Link
Anti-LilrB2	Mouse monoclonal		Cytometry: Immunofluorescence:	http://www.abcam.com/lilrb2-antibody-ab172538.html
Anti-PirB	Goat 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.

Transfection Plan:

LilrB2

Expand

title	Trial 1

Expand

title	Microscopy 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

title	Cytometry 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

PirB

Expand

title	Trial 1

Expand

title	Microscopy 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

title	Cytometry 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

Expand

title	Trial 2

Expand

title	Microscopy 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

title	Cytometry 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

Status:

First trial with PirB done
First trial with LilrB2 done

Results:

Expand

title	PirB Trial 1

Microscopy


Control	Permeabilized	Non-permeabilized	LilrB2 Paper

Cytometry

Expand

title	Gating

SSC-A vs FSC-A	FSC-H vs FSC-W	SSC-H vs SSC-W	Yellow vs Red

Tube 1	Tube 2	Tube 3	Tube 4

Tube 5	Tube 6	Tube 7	Tube 8

Expand

title	LilrB2 Trial 1

Microscopy

Permeabilized	Non-permeabilized	Non-permeabilized stained control

Cytometry

Tube 2	Tube 3	Tube 4	Tube 5



Tube 8	Tube 9	Tube 10	Tube 11

Overlay of 4 and 5:

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

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Versions Compared

Old Version 77

New Version 78

Key

Description:

Cloning

Description:

Plan:

Status:

Plan:

Experiment

Description:

Transfection Plan:

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