Malinow: Difference between revisions
Bradley Monk (talk | contribs) No edit summary |
Bradley Monk (talk | contribs) No edit summary |
||
Line 1: | Line 1: | ||
==Proteins that interact with AMPARs== | ==Proteins that interact with AMPARs== | ||
Line 39: | Line 41: | ||
==NRSA== | ==NRSA== | ||
* Dominant negative [[PSD95]] | * Dominant negative [[PSD95]] | ||
==Choquet Email== | |||
Hi Roberto, | |||
I hope you’re doing well, haven’t seen each other in a while. As far as receptor tracking in slices go, we’ve not progressed much. As you’ve done, we routinely use FRAP of phluorin-tagged receptors to evaluate mobility in slices, and this works well, except for the over-expression issue. | |||
As for quantum dot tracking in slices, our own trials have been quite unsuccessful, most QDs being generally too sticky and not diffusing well in tissue. Thus, as for tracking endogenous receptors, I think it’s quite hopeless. I do have seen in a few other labs people using GFP tagged proteins and managing to track them with anti-GFP coated QDs, but I have no direct experience with this approach as if I’m to use a tagged receptor, I prefer then to use FRAP in slice as it’s less prone to artifacts I think. | |||
Sorry I can’t [[help]] more, sure I’d wish we could do that…… | |||
All the best and see you in the near future | |||
Best | |||
Daniel |
Revision as of 16:21, 3 July 2013
Proteins that interact with AMPARs
Qdots
Getting a Qdot into the cell
- Conjugate Qdot with secondary antibody fab
- Incubate tissue with primary antibodies for AMPAR and PSD95
- Puff Qdots onto cell body, these will bind the primary at AMPAR N-terminus
- When AMPARs internalize the Qdot will be dragged into cell
- Cleave N-terminus of AMPAR to liberate Qdot
- Qdot can then bind the primary ligated to PSD95
Notes
- Molecular Methods
- FLASH technology
- Bredt
- minisog - gfp
- Acidic basic polypeptide recognition sequences
- Talk with nanotech group about various ways to conj. Qdots
- Nichol and England - couple Qdot to AMPAR agonist
- Have simulation be a competitive model where AMPARs are competing during LTP
- Quantitative review on synaptic numbers (Sheng)
PALM STORM
There are two major groups of methods for functional super-resolution microscopy:
1. Deterministic super-resolution: The most commonly used emitters in biological microscopy, fluorophores, show a nonlinear response to excitation, and this nonlinear response can be exploited to enhance resolution. These methods include STED, GSD, RESOLFT and SSIM.
2. Stochastical super-resolution PALM STORM: The chemical complexity of many molecular light sources gives them a complex temporal behaviour, which can be used to make several close-by fluorophores emit light at separate times and thereby become resolvable in time. These methods include SOFI and all single-molecule localization methods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM and dSTORM.
NRSA
- Dominant negative PSD95
Choquet Email
Hi Roberto,
I hope you’re doing well, haven’t seen each other in a while. As far as receptor tracking in slices go, we’ve not progressed much. As you’ve done, we routinely use FRAP of phluorin-tagged receptors to evaluate mobility in slices, and this works well, except for the over-expression issue. As for quantum dot tracking in slices, our own trials have been quite unsuccessful, most QDs being generally too sticky and not diffusing well in tissue. Thus, as for tracking endogenous receptors, I think it’s quite hopeless. I do have seen in a few other labs people using GFP tagged proteins and managing to track them with anti-GFP coated QDs, but I have no direct experience with this approach as if I’m to use a tagged receptor, I prefer then to use FRAP in slice as it’s less prone to artifacts I think. Sorry I can’t help more, sure I’d wish we could do that……
All the best and see you in the near future
Best
Daniel