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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 (Red) |
Well 1 HEK293 | Well 2 HEK293 Dummy DNA eYFP Streptavidin | Well 3 HEK293 Dummy DNA eYFP 1000 nM Monomerized aBeta Streptavidin | Well 4 HEK293 Dummy DNA eYFP 1000 nM Oligomerized aBeta Streptavidin | Well 5 HEK293 Gmab H Gmab L CD79A CD79B eYFP Streptavidin | Well 6 HEK293 Gmab H Gmab L CD79A CD79B eYFP 1000 nM Monomerized aBeta Streptavidin |
Well 7 HEK293 Gmab H Gmab L CD79A CD79B eYFP 1000 nM Oligomerized aBeta Streptavidin | Well 8 EMPTY | Well 9 EMPTY | Well 10 EMPTY | Well 11 EMPTY | Well 12 EMPTY |
Well 13 EMPTY | Well 14 EMPTY | Well 15 EMPTY | Well 16 EMPTY | Well 17 EMPTY | Well 18 EMPTY |
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 | Well 3 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP 0.25 nM abeta oligomer | Well 4 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP 0.5 nM abeta oligomer | 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 | Well 9 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP 25 nM abeta oligomer | Well 10 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP 50 nM abeta oligomer | 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 |
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 that saturates at approximately 100 nM abeta based on the specifications of Gantenerumab. We are unsure whether the antibody will bind both monmerized and oligomerized beta amyloid.
(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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