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

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

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

Well 1

HEK293

Well 2

HEK293

Dummy DNA

Well 3

HEK293

hEF1a: eYFP

Dummy DNA

Well 4

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP


Well 5

HEK293

1000 nM abeta

Well 6

HEK293

Dummy DNA

Anti IgM

1000 nM abeta

Well 7

HEK293

hEF1a: eYFP

Dummy DNA

1000 nM abeta

Well 8

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

1000 nM abeta

 EMPTYEMPTYEMPTY

Well 13

HEK293

hEF1a: GmabL

hEF1a: GmabH

hEF1a: CD79A

hEF1a: CD79B

hEF1a: eYFP

0 nm abeta

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

 

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

 

 

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