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Well 8

HEK293

Dummy DNA

hEF1a: eYFP

Well 9

HEK293

Dummy DNA

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

Expand
titleTest1
Expand
titleDescription

In this experiment, we will determine the affinity of beta-amyloid for the variable regions of our mIgM in the B-cell receptor.

Expand
titlePurpose

We plan to determine whether beta-amyloid binds to our BCR. We would also like to establish a Kd value if possible.

Expand
titleParts Needed
Parts
pEXPR hEF1a: Gmab Heavy
pEXPR hEF1a: Gmab Light
pEXPR hEF1A: CD79A+stop
pEXPR hEF1a: CD79B+stop
pEXPR hEF1a: eBFP
anti-IgM
beta amyloid
streptavidin-fluorophore (Red)
Expand
titleSetup

 

 

Well 1

HEK293

Well 2

HEK293

Dummy DNA

hEF1a:

eYFP

Streptavidin

Well 3

HEK293

Dummy DNA

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

Well 5

HEK293

eYFP

500 nM Monomerized aBeta

Streptavidin

Well 4

HEK293

 

Dummy DNA

hEF1a: eYFP

eYFP

500 nM Oligomerized aBeta

Streptavidin

Well

6

5

HEK293

Dummy DNA

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

Well 7

HEK293

Well 10

HEK293

Gmab H

Gmab L

CD79A

CD79B

eYFP

Streptavidin

Well 6

HEK293

Gmab H

Gmab L

CD79A

CD79B

eYFP

500 nM Monomerized aBeta

Streptavidin

Well 7

HEK293

Gmab H

Gmab L

CD79A

CD79B

eYFP

500 nM Oligomerized aBeta

Streptavidin

Well 8

EMPTY

Well 9

EMPTY

Well 10

EMPTY

Well 11

EMPTY

Well 12

EMPTY

Well 13

Well 11

HEK293

Dummy DNA

hEF1a: eYFP

Well 12

HEK293

Dummy DNA

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

Well 13

HEK293

Well 14

HEK293

Dummy DNA

hEF1a:

eYFP

Streptavidin

Well

15

14

HEK293

Dummy DNA

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

eYFP

Streptavidin

Beta-amyloid oligomers 500nM

Well 15

HEK293

hEF1a: eYFP

Well 16

HEK293

Streptavidin

 

Well 17

HEK293

Dummy DNA

Streptavidin

aBeta Oligomers

hEF1a: eYFP

Well 18

HEK293

Dummy DNA

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

Streptavidin

Beta-amyloid oligomers 500nM

Well 19

EMPTY

Well 20

EMPTY

Well 21

EMPTY

Well 22

EMPTY

Well 23

EMPTY

Well 24

EMPTY

 

 

 

Well 1

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

0 nM abeta oligomer

 

Well 2

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

0.1 nM abeta oligomer

Streptavidin

Well 3

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

0.25 nM abeta oligomer

Streptavidin

Beta-amyloid Oligomers 500nM

Well 4

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

0.5 nM abeta oligomer

Streptavidin

Beta-amyloid oligomers 500nM

Well 5

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

1 nM abeta oligomer

 

Well 6

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

2.5 nM abeta oligomer

 

 

Well 7

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

5 nM abeta oligomer

 

Well 8

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

10 nM abeta oligomer

Streptavidin

Well 9

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

Streptavidin

Beta-amyloid monomers

25 nM abeta oligomer

Well 10

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

50 nM abeta oligomer

Streptavidin

Beta-amyloid oligomers

Well 11

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

100 nM abeta oligomer

 

Well 12

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

1000 nM abeta oligomer

 

Well 13

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

0 nM abeta oligomer

Well 14

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

.1 nM abeta

Well 15

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

.25 nM abeta

Well 16

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

.5 nM abeta

Well 17

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

1 nM abeta

Well 18

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

2.5 nM abeta

Well 19

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

5 nM abeta

Well 20

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

10 nM abeta

Well 21

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

25 nM abeta

Well 22

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

50 nM abeta

Well 23

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

100 nM abeta

Well 24

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

1000 nM abeta
Expand
titleExpected Results

We expect to find clear membrane localization of red alexafluor conjugated streptaviding bound to beta amyloid bound to the BCR antibody. This will give us red halos around yellow cells with potentially blue nuclei due to DAPI staining. Moreover we expect a Kd on the order of 1 nM abeta based on the specifications of Gantenerumab. (According to the paper which introduced it, the Kd was 0.6 for plaques, 1.2 for oligomers, and 17 for monomers.) We are unsure whether the antibody will bind both monmerized and oligomerized beta amyloid.

Expand
titleProtocol

 

 

(http://www.renyi.hu/~stipsicz/skin/Flow_cytometry_book/Ch_17.pdf  p.16-19 - protocol for yeast using c-myc as transfection marker)

Expand
titleData

 

Expand
titleAnalysis

 

 

 

Expand
titleTest2
Expand
titleDescription

In this experiment, we will determine the affinity of beta-amyloid for the variable regions of our mIgM in the B-cell receptor.

Expand
titlePurpose

We plan to determine whether beta-amyloid binds to our BCR. We would also like to establish a Kd value if possible.

Expand
titleParts Needed
Parts
pEXPR hEF1a: Gmab Heavy
pEXPR hEF1a: Gmab Light
pEXPR hEF1A: CD79A+stop
pEXPR hEF1a: CD79B+stop
pEXPR hEF1a: eBFP
anti-IgM
beta amyloid
streptavidin-fluorophore (Red)
Expand
titleSetup

Well 1

HEK293

Well 2

HEK293

eBFP

Dummy

Well 3

HEK293

eBFP

Dummy

aBeta

Strep

Well 4

HEK293

eBFP

Gmab H

Gmab L

CD79A

CD79B

Strep

Well 5 

HEK293

Gmab H

Gmab L

CD79A

CD79B

eBFP

aBeta

Strep

Well 6 

EMPTY

Well 7

HEK293

Well 8 

HEK293

eBFP

Dummy


Well 9 

HEK293

eBFP

Dummy

aBeta

Strep

 

Well 10 

HEK293

eBFP

Gmab H

Gmab L

CD79A

CD79B

Strep

Well 11 

HEK293

Gmab H

Gmab L

CD79A

CD79B

eBFP

aBeta

Strep

Well 12 

HEK293

Well 13 

HEK293

Well 14 

HEK293

Well 15 

HEK293

Well 16 

HEK293

Well 17 

HEK293 

Well 18 

HEK293

Well 19 

HEK293

Well 20

HEK293

Well 21 

HEK293

Well 22 

HEK293 

Well 23 

HEK293

 

Well 24 

HEK293

Expand
titleExpected Results

We expect to find clear membrane localization of red alexafluor conjugated streptaviding bound to beta amyloid bound to the BCR antibody. This will give us red halos around yellow cells with potentially blue nuclei due to DAPI staining. Moreover we expect a Kd on the order of 1 nM abeta based on the specifications of Gantenerumab. (According to the paper which introduced it, the Kd was 0.6 for plaques, 1.2 for oligomers, and 17 for monomers.) We are unsure whether the antibody will bind both monmerized and oligomerized beta amyloid.

Expand
titleExpected Results
 

Expand
titleProtocol

 

 

(http://www.renyi.hu/~stipsicz/skin/Flow_cytometry_book/Ch_17.pdf  p.16-19 - protocol for yeast using c-myc as transfection marker)

Expand
titleData
Expand
titleGates

Image Added Image Added Image Added

UntransfectedeBFP

Image Added

 

Image Added
eBFP + streptavidin + AbetaBCR + streptavidin
Image Added

Image Added

 

BCR + streptavidin + Abeta 
Image Added 
Expand
titleAnalysis