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Description:
In this experiment, we are aiming to test whether or not our receptor proteins localize to the cell membrane. To do that, we will have HEK293 cells express LilrB2 and PirB. We will test for membrane localization of the receptors through immunofluorescence. We will use primary antibodies that will bind to LilrB2 and and PirB and secondary antibodies conjugated to fluorophores that will bind to the primary antibodies.
Cloning
COMPLETE
Description:
Making pEXPRs hEF1a:LilrB2 and hEF1a:PirB.
Plan:
- Order LilrB2 and PirB gBlocks
- Simulate Golden Gate on Geneious and make sure it works
- 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
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Status:
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Experiment
Description:
Testing membrane localization of receptor proteins using anti-LilrB2 and anti-PirB primary antibodies and secondary antibodies conjugated to yellow Alex Fluors.Alexa Fluors.
Antibody | Type | Conjugation | Dilution Used | Link |
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| Anti-LilrB2 | Mouse monoclonal | Cytometry: Immunofluorescence: | http://www.abcam.com/lilrb2-antibody-ab172538.html | |
| Anti-PirB | Goat polyclonal | Cytometry: Immunofluorescence: | http://www.scbt.com/datasheet-9609-Pirb-c-19-antibody.html | |
Donkey Anti-Goat IgG | Alexa Fluor 488 | Cytometry: 1/2000 Immunofluorescence: 1/200 - 1/1000. | ||
| Donkey Anti-Mouse IgG | Alexa Fluor 488 | Cytometry: 1/2000 Immunofluorescence: 1/200 - 1/1000. |
Transfection Plan:
LilrB2
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PirB
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Status:
- First trial with PirB done
- First trial with LilrB2 done
Results:
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Microscopy
Cytometry
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Cytometry
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Overlay of 4 and 5:
| hef1a:mKate + dummy | hef1a:mKate + hef1a:LilrB2 | |||
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