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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 Streptavidin | Well 3 HEK293 1000nM Streptavidin | Well 4
| Well 5
| Well 6
|
Well 7
| Well 8
| Well 9
| Well 10
| Well 11
| Well 12
|
Well 13
| Well 14
| Well 15
| Well 16
| Well 17
| Well 18
|
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 |
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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