Description: |
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| 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. |
CloningCOMPLETE Description:Making pEXPRs hEF1a:LilrB2 and hEF1a:PirB.
| Plan:
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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 Alexa Fluors.
Antibody | Type | Conjugation | Dilution Used | Link |
|---|---|---|---|---|
| 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. |
Results:
Microscopy
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| Control | Permeabilized | Non-permeabilized | LilrB2 Paper |
Cytometry
| SSC-A vs FSC-A | FSC-H vs FSC-W | SSC-H vs SSC-W | Yellow vs Red |
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| Tube 1 | Tube 2 | Tube 3 | Tube 4 |
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| Tube 5 | Tube 6 | Tube 7 | Tube 8 |
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Microscopy
| Permeabilized | Non-permeabilized | Non-permeabilized stained control | |
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Cytometry
| Tube 2 | Tube 3 | Tube 4 | Tube 5 |
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| Tube 8 | Tube 9 | Tube 10 | Tube 11 |
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Overlay of 4 and 5:
| hef1a:mKate + dummy | hef1a:mKate + hef1a:LilrB2 | |||
| + | | = |  |