Qs: pathogenic form 맞니? 교수? 더 확인없이 csf 기도 되니? How to …
- Co-IP?
- Antibody: purchase particularly for ASC, GSDMD
- To add: mouse, rat, brain/CSF/Plasma
- Should be a little complex than single molecular enzyme detection (Cosomil) (sample preparation: CSF detergent, larger volume:
Pablo Pelegrin, Roche (2 times, biofluid)
| Active (Speck-bound) NLRP3 | Active (speck-bound) ASC | Active caspase-1 | GSDMD (N-terminal domain, cleaved) | |
|---|---|---|---|---|
| GFP-NLRP3/mCh-ASC Speck detection in THP-1 is still in progress |
GFP-NLRP3/mCh-ASC Speck • THP-1 (PMA + Nigericin) • MM PBMCs Co-IP? |
GFP-Caspase-1 FL/p20 speck-bound IF (=) Co-IP? |
GFP-GSDMD FL/NTD MMP THP-1, MultiBO X-1 (THP-1) MM PBMC, • active GSDMD (THP-1 & MM PBMC) | |
| Validation? | PMA/Nigericin → ↑ (THP-1), |
inhibition by MCC950 (THP-1) ↑ il-1b (THP-1) ∝ active GSDMD (thp-1 & MM PBMC) | Co-IP? | |
| Prof. Juan Pablo de Rivero Vaccari (Univ. of Miami) measured ASC specks from CSF samples of HD and AD patients through Western linked ASC oligomer. ASC speck levels are detected at higher levels in AD CSF compared to HD ※ data X publication ([Vontell, #2250) X | ||||
| verify | Tsd_(GFP-labeled IF): Proteina also us Shipment by frozen vs fixed? | |||
| Mouse | ||||
Rodent: mouse/rat 6 pooled → 10 indivisual?
- human: directly individual (분제 X 그러나 cost sample & service 고
- Mouse: characterize (assay requires 100ul, so it would take at least 10 mouse data point.)
- normal mouse (individual 도 bioIVT 에서 가능한 듯)
- disease model samples: Atuka: too precious, (2w) bzATP mouse (CSF, br…)
- validation study (Takeda plan → pass to Proteina),
- Minimum: 10 uL
Proteina Bob. Scienist.com: 48D6FD: Assay development: NLRP-INFLMMASOME
Pyroptosis involves the formation of NLRP3 (NLR Family Pyrin Domain Containing 3)-inflammasome. The inflammasome is a multi-protein complex consisting of oligomerized NLRP3 and ASC protein. ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) binds with the altered NLRP3 and assembles into multimers. There is a need of the development of a novel biochemical assay that can sensitively measure the NLRP3-ASC speck complex in human/rodent biofluid (CSF and plasma). Commercial assays are already available for ASC protein and we are not interested in developing an assay to individually measure NLRP3 or ASC protein.
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Pyroptosis involves the formation of NLRP3 (NLR Family Pyrin Domain Containing 3)-inflammasome. The inflammasome is a multi-protein complex consisting of oligomerized NLRP3 and ASC protein. ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) binds with the altered NLRP3 and assembles into multimers. There is a need to sensitively measure the quantity of the NLRP3-ASC speck complex in human biofluid (CSF and plasma) by a novel biochemical assay. We are looking for an assay that has already been developed and optimized for the analyte so human biofluid samples can be tested. Commercial assays are already available for ASC protein on its own and we are not interested in developing assays for ASC protein that exist individually outside of the inflammasome.
| NLRP3 | ASC | GSDMD | ||
|---|---|---|---|---|
| Human | Detect what it intends to detect? |
- ↑ by Nigericine (THP1 cell pellet & media) - no Titration curve yet - GFP-NLRP3 |
- ↑ by Nigericine (THP1 cell pellet & media) - Titration curve O |
- ↑ by Nigericine (THP1) cell pellet & media) - ↑ eGFP-labelled NTD (vs FL) GSDMD (HEK293T) |
| Pharmac modulation? | ↓ by MCC950 (cs50 19.8 nM in THP1) | |||
| 대체성? |
[Inflammasome: NLRP3 oligomerized] -NLRP3 S-plex: monomer 아닌가? |
[Inflammasome: ASC] -Otake's M speck! -Flow cytometry: fluorophore distribution -Klenerman: ASC speck IF (=) (?) |
Otake: FL Quanterix SIMOA: NTD (validation stage, 어느 기다 둘만?) SIMOA: 만 들어본 적 있고 Proteina 가 co-IP 이니 X 일까? | |
| PBMC | 73% were minimal level (without stimulation), so no window expected. | 63% were minimal level (without stimulation), so no window expected. | ||
| Mouse | Detect what it intends to detect? |
- Detected in J774A.1 cells (active form → ↑ by LPS (THP1), - 20230602 Mouse active NLRP3 assay development is currentl in progress |
- Detected in J774A.1 cells (in both cells and media), (Active form induced could be induced by LPS priming & Nigericin (20230602) (not detected by NASC assay) - Plan: to determine sensitivity | |
| Pharmac modulation? | ||||
| Rat | Detect what it intends to detect? | |||
| Pharmac modulation? | ||||
[comparison]
Fibril: clump together,
| (Proteina) Co-IP | WB | Mass spec | Pull-down (Cambridge) | ||
|---|---|---|---|---|---|
| 되짚아 My experence | |||||
| Native vs denatured | Denatured | Denatured (boiled) | Detergents can be denaturing or non-denaturing | Native (Jain 2019) | |
| Method to detect insoluble proteins | Expression & purification | (Trinpus 2009) Solvent-free sample preparation and ionization: detergent 가 insoluble protein 을 녹임으로써, solubilize protein 이라 | |||
| Analysis | Blue native gel electrophoresis in which protein complexes are resolved in native form in the first dimension prior to denaturing SDS-PAGE in the second dimension (17). | (Trinpus 2009) Fusion tags (His) rendering the fusion protein soluble. E. coli free system, microfluidic device, co-expression of chaperones | |||
detergents are used to lyse cells (solubilizing lipids in the cell membrane and releasing proteins), solubilize membrane proteins and lipids, control protein crystallization, prevent non-specific binding
| Proteina | Charles River | |
|---|---|---|
| There are some reports that active ASC (ie inflammasome-bound) takes on fibrillar form. Can Proteina's Co-IP method detect insoluble proteins? If so, how? |
|
largely should depend on whether antibody recognize insoluble AG, Denature-dilute-capture, is the ASC in inflammasome soluble (fibril, possibly)? Will use → Pre-treatment: NLRP3 / protein (-) → ASC / protein on its (-) |
| how they make this assay quan... | ||
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
Pablo Pelegrin sub-table / column placement | the Active (Speck-bound) NLRP3 / Active (speck-bound) ASC / Active caspase-1 / GSDMD columns are inferred from the visible cell text in body_r01_c01.jpg; column boundaries are partly clipped at the right edge. | low confidence on column-by-cell placement. |
Mouse / characterize bullet / disease model samples | reads (2w) bzATP mouse (CSF, br...) with br... clipped at the cell edge (likely brain); preserved verbatim. | clipped; partial. |
Per-marker sub-table / 대체성? row / NLRP3 cell | reads [Inflammasome: NLRP3 oligomerized] -NLRP3 S-plex: monomer 아닌가?; the S-plex token is preserved verbatim. | low confidence on whether S-plex denotes Quanterix S-plex or a different platform. |
Charles River column / Pre-treatment line | reads Will use → Pre-treatment: NLRP3 / protein (-) → ASC / protein on its (-) as a workflow note; preserved verbatim. | low confidence on whether the (-) markers denote negation or empty cells. |