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titleTest2
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titleDescription

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

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titlePurpose

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

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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)
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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

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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.

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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)

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titleData
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titleGates

 

 

UntransfectedeBFP

 

eBFP + streptavidin + AbetaBCR + streptavidin

 

BCR + streptavidin + Abeta 
 
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titleAnalysis

 

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