- Created by Unknown User (clrichar@mit.edu), last modified by Unknown User (krbrink@mit.edu) on Aug 08, 2014 16:17
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 500 nM Monomerized aBeta Streptavidin | Well 4 HEK293 Dummy DNA eYFP 500 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 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 HEK293 Dummy DNA eYFP Streptavidin | Well 14 HEK293 Dummy DNA eYFP Streptavidin Beta-amyloid oligomers 500nM | Well 15 HEK293 | Well 16 HEK293 Streptavidin
| Well 17 HEK293 Streptavidin aBeta Oligomers | Well 18 HEK293 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
| Well 2 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP Streptavidin | Well 3 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP Streptavidin Beta-amyloid Oligomers 500nM | Well 4 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP Streptavidin Beta-amyloid oligomers 500nM | Well 5 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP
| Well 6 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP
|
Well 7 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP
| Well 8 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP Streptavidin | Well 9 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP Streptavidin Beta-amyloid monomers | Well 10 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP Streptavidin Beta-amyloid oligomers | Well 11 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP
| Well 12 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP
|
Well 13 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 14 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 15 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 16 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 17 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 18 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP |
Well 19 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 20 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 21 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 22 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 23 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP | Well 24 HEK293 hEF1a: GmabL hEF1a: GmabH hEF1a: CD79A hEF1a: CD79B hEF1a: eYFP |
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.
(http://www.renyi.hu/~stipsicz/skin/Flow_cytometry_book/Ch_17.pdf p.16-19 - protocol for yeast using c-myc as transfection marker)
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 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 |
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.
(http://www.renyi.hu/~stipsicz/skin/Flow_cytometry_book/Ch_17.pdf p.16-19 - protocol for yeast using c-myc as transfection marker)
| Untransfected | eBFP |
|---|---|
|  |
| eBFP + streptavidin + Abeta | BCR + streptavidin |
 |
|
| BCR + streptavidin + Abeta | |
 |
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