Description:
In this group of experiments, we are aiming to test the affinity of our receptors, LilrB2 and PirB to beta-amyloid oligomers. To do that, we will have HEK293 cells express LilrB2 and PirB fused with YFP. We will then apply biotinylated beta-amyloid oligomers to the LilrB2 and PirB expressing cells. To test for binding, we are going to use the biotin-binding protein streptavidin covalently bonded to a fluorophore. We will look for co-localization of the fluorophore and the beta-amyloid.
*We may potentially extend this experiment to trying to characterize the interaction between beta-amyloid and the receptors i.e. try to generate saturation plots and calculate dissociation constants.
Cloning
Description:
Make HEK293 expressing LilrB/PirB - YFP fusions. We will test binding beta-amyloid in Experiment B2
Plan:
- Simulate PCR and Golden Gate on Geneious and make sure it PCR product will be compatible with eYFP and TCS-Gal4VP16 PCR products from other subgroups
- Golden Gate gBlocks into ggDONR
- Transform bacteria with Golden Gate product and plate
- Pick colonies and grow in liquid culture
- Miniprep cells and verify product
- Make glycerol stocks
- LR into pDEST
- Transform, pick colonies and grow in liquid culture
- Miniprep cells and verify product
- Make glycerol stocks
- Midiprep product
Status:
- gel run to verify PCR products
- LilrB2 did not give expected band size
- why?
- PirB did not give expected band size
- pENTR used as template was not right
- LilrB2 did not give expected band size
- verification gel to be run
Experiment
Description:
Testing beta-amyloid binding to receptor proteins using biotinylated oligomerized beta-amyloid. We will test for binding using he biotin-binding protein streptavidin covalently bonded to a fluorophore.
Transfection Plan:
LilrB2
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PirB
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Status:
Not yet started.
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Results
LilrB2
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Transfection marker: tagBFP Staining: biotinylated beta amyloid and strep-AF594
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PirB
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Analysis
LilrB2
Trial 1:
The cell population transfected with LilrB2 does not show any increase in red fluorescence which may indicate that LilrB2 does not bind abeta. However, from the picture taken of the cells before staining, it appears that the transfection efficiency for that well is very low making the reliability of the results form this trial very questionable.
PirB
Cytometry Trial 1:
The cell population transfected with PirB shows a marked increase in red fluorescence which indicates that PirB is in fact binding abeta. Moreover, when the distribution of red fluorescence of the transfected population (gated using the blue fluorescence distribution) is plotted, you see a shift in the distribution towards more red fluorescence and the mean increases from 171 in the PirB - population to 1069 in the PirB + population.
It is interesting to note the "non-linear" distribution of red vs. blue in the PirB + population. We do not begin to see a significant increase in red fluorescence until we reach the more highly transfected cells. It would be interesting to plot mean blue vs. mean red to try to analyze this further.
- Next steps: Microscopy! to try and confirm the results we are getting through cytometry