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BCR=CD79A+CD79B+IgM heavy+IgM light
| EXP # | blocked by(do this first) | Condition for performing | What | Why | Parts | Basic procedure | Input (independent variable being tested) | Expected Output (dependent variable being measured, preferably with meaning and failure modes) | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| -1 | none | Western blot cells for Syk and Lyn | to determine whether they already express these proteins | anti-Syk, anti-Lyn, secondary antibodies | Western blot HEK293 cells | |||||||
0 | none (maybe 7) | Testing is for debug | Point mutate Syk to keep it from being phosphorylated | No phosphorylation = no downstream effects | Syk plasmid | Put mutated and unmutated versions in cell with constitutive promoter and assay for phosphorylation | Neither should phosphorylate (this is concerning that control output = experimental, is this a test we do later? Do we not test point mutations and trust that they work) | |||||
| 1 | none | Testing is for debug | Point mutate Lyn to make it autophosphorylate | Control: Even if the BCR complex does not work (eg doesn't detect amyloid), we should still see Syk recruitment | Lyn plasmid | Put mutated and unmutated versions in cell with constitutive promoter and assay for phosphorylation | normal should not phosphorylate, mutated version should be phosphorylated. | |||||
| 2 | none | Just practice | Check tev cleavage system in general | |||||||||
| 3 | none | only 3 or 4 needs to be done, 4 tells localization (cytosol or membrane) 3 only says membrane or not. | Check IgM localization using cytometry (see Hombach paper) (is this needed, if CD79 localizes, we are pretty sure IgM had to localize correctly) | IgMkappa IgM heavy CD79a CD79b | add all CD79A, CD79B, heavy chain, light chain all untagged. Add stained antibody that targets igM (heavy and/or light?) and see if it sticks to the cell membrane. (add and wash) | test 1: CD79A/B and one test for each leader sequence used, stained igM targeting antibody control: HEK cells, no BCR circuit | test1: outside of cell glows = correct localization cell doesn't glow = bad localization, failure to express, or antibodies secreted. control: no glow anywhere | |||||
| 4 | none | 3 | debug for 3, shows cytosol vs membranesame | Check CD79a/b localization (we have to have a CD79 fused to an FP for the final product, so might as well test localization now) | fluorescently tagged CD79a/b | add all BCR components, with one CD79 at a time in its FP tagged version to check correct localization | test 1: CD79A-tev-FP, CD79B, igM test 2: CD79B-tev-FP, CD79A, igM | test 1: cell membrane glows: correct localization. Cytosol glows = not membrane localized. No glow = not produced test 2: same, but different tested protein | ||||
| 5 | 3/4 | only if membrane localization fails, may test why | Test that things stick together (CD79A/B+IgM heavy and light stick together (coIP)(make this test description not suck) | BCR, IgM heavy and light | do some gel tests | tests for components | all together should move slowly through gel | |||||
| 6 | 3 | Check IgM binds to beta amyloid(talk to Brian) | ?, antibodydo some kind of a wash step to see if it sticks? Express antibody, lyse cells and wash over beta amyloid, wash with a dyed IgM targeting antibody. | This test needs to be updated | one test for each antibody with dyed antibody , andthat localizes correctly | control should not glow Glow should mean that dyed antibody didn't wash off because it stuck to IgM and IgM didn't wash off because it stuck to beta amyloid lack of glow in test means either the antibody doesn't bind to amyloid or that our test is borked (we could try to control for a bad test by using an antibody that we know works that doesn't bother with the membrane). | ||||||
| 7 | 3/4 | Check Lyn localization | BCR Lyn-GFP | Add all BCR not FP tagged, add FP tagged Lyn, see what glows | test 1: BCR, lyn control: no BCR, lyn | cell membrane glows: correct localization Cytosol glows: not binding to CD79 no glow: not being expressed control: cytosol should glow? | ||||||
| 8 | 7 | Check Syk localization | Syk-GFP, BCR, beta amyloid | add all BCR and get Lyn phosphorylated, and FP tagged Syk If lyn autophosphorylation doesn't work, use IgM | test 1(control): normal Lyn test 2: autophsosphorlated Lyn | test 1: Syk should float in cytosol test 2: Syk should localize to membrane (if test 2 fails, it my be because the localization is too weak to test, so we should use non mutant syk + tev and assay for phosphorylation)
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| 9 | 8 | debug if 8 fails (if the membrane localization is too weak, FP won't distinguish) consider doing instead of 7 | Check BCR function (does syk/lyn phosphorylate) | BCR, Lyn, Syk, phosphorylation assay | run with and without BCR and beta amyloid and assay for phosphorylation | no beta, no bcr bcr, no beta amyloid bcr, beta amyloid | 1: no phosphorylation 2:no phosphorylation 3:lyn and syk phosphorylated | |||||
| 10 | 8, (9 if we use beta amyloid to activate Lyn, just 8 if we use anti IgM) | Syk+TEV (variableon a dox inducible promoter (also a mKate with same promoter to be sure we know concentration) Heavy chain-FP, with and without TEV- transcriptional activator (TA) Light chain beta-amyloid Lyn-autophosphorylation and amyloid and anti IgM (we need an active and inactive form) CD79-TEVsite-FP, with and without TEV-TA Inducible activator and eBFP to measure output | add all components and syk+tev, lyn is active | test: every combination of normal and FP tagged CD79A/B and FP tagged IgM with active lyn control: same, but lyn inactive | For every combination (Lyn inactive however), test several logarithmically distributed concentrations of dox to find what syk expression rate is to high For every combination (Lyn active however), test several logarithmically distributed concentrations of dox to find what syk expression rate is to low pick an in between value which has the best on/off difference of expression | Control: fps on membrane test group: some of fps move to cytosol compared to control | ||||||
| 11 | 10,9 | 12 failed | Check that beta amyloid causes syk recruitment (may be just to debug if the whole system check fails, if whole system check passes, we don't need this) | Syk-FP CD79 not tagged IgM Lyn, normal | assemble cells, measure Syk localization as a function of beta amloid | Vary beta amyloid (either continuum or none and a lot or none and a few levels). Do one test for each leader sequence. | Should get a curve of how much syk localizes to the membrane as a function of beta amyloid concentration. | |||||
| 12 | 10, 9 | If test 10 uses beta amyloid to activate Lyn, this is a duplicate | Check whole system | CD79-TEV-FP igM heavy and light Lyn circuit and Syk-TEV concentration selected from test 10beta amyloid (variable) | add CD79A/B (with FP tag from tev cleavage test), Lyn, syk+TEV, and one test for every leader sequence that works. Add some amount of beta amyloid and get a curve of FP cleavage to beta amyloid (or, if knowing beta amyloid concentration is hard, we should still characterize something so that future researchers can tune the amount of output). | for each antibody leader: add components, add beta amyloid (there should be a control with 0 beta amyloid, I'd like to have varying amounts of beta amyloid) | Some amount of FP should move from membrane to cytosol (either compare to control of no amyloid or compare intensity of membrane to intensity of cytosol) | Not sure what this should do yet, may just test one circuit, may test every binding and localizing antibody, may try to vary beta amyloid concentration to see what our reporter as a function of beta amyloid concentration looks like. |
Supplies needed
Proteins needed
Supplies needed
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Things to test:
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- does antibody get to membrane?
- does cd79A/B get to membrane?
- does lyn attach?
- does syk get recruited by lyn?
- ? test CD79x dimerization with gel
- ?test CD79x-IgM attachement with gel
...