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AVANTI POLAR LIPIDS , CYGNUS , LARODAN , ADIPOGEN , MYBIOSOURCE , SERVA NORDMARK, BIOWORLD, EDGEBIO , PANREAC APPLICHEM
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NEW NLRP3 Inflammasome Starter Sets - NOW AVAILABLE !
| PID | PRODUCT NAME | PROTOCOL |
| AG-44B-0008 |
NLRP3 Inflammasome Human Antibodies Starter Set Manual |
The NLRP3 Inflammasome Human Antibodies Starter Set is an all-in-one solution to study the NLRP3 inflammasome using Western blotting application. This antibody starter set comprises our KO extract validated STANDARD antibodies against the key components of the NLRP3 inflammasome including NLRP3, Asc and Caspase-1, used and published by the experts in inflammasome research. This economic starter set contains enough primary antibodies to perform at least 3 western blot experiments. |
| AG-44B-0009 |
NLRP3 Inflammasome Mouse Antibodies Starter Set Manual |
The NLRP3 Inflammasome Mouse Antibodies Starter Set is an all-in-one solution to study the NLRP3 inflammasome using Western blotting application. This antibody starter set comprises our KO extract validated STANDARD antibodies against the key components of the NLRP3 inflammasome including NLRP3, Asc and Caspase-1, used and published by the experts in inflammasome research. This economic starter set contains enough primary antibodies to perform at least 3 western blot experiments. |
| AG-44B-0010 |
NLRP3 Inflammasome Human Reagents Starter Set Manual |
The NLRP3 Inflammasome Human Reagents Starter Set is an all-in-one solution to study the NLRP3 inflammasome using Western blotting application. This reagents starter set comprises our KO extract validated STANDARD antibodies against the key components of the NLRP3 inflammasome including NLRP3, Asc and Caspase-1, used and published by the experts in inflammasome research. It also contains two chemical compounds for priming and activation of the NLRP3 inflammasome. This economic starter set contains enough primary antibodies to perform at least 3 western blot experiments. |
| AG-44B-0011 |
NLRP3 Inflammasome Mouse Reagents Starter Set Manual |
The NLRP3 Inflammasome Mouse Reagents Starter Set is an all-in-one solution to study the NLRP3 inflammasome using Western blotting application. This reagents starter set comprises our KO extract validated STANDARD antibodies against the key components of the NLRP3 inflammasome including NLRP3, Asc and Caspase-1, used and published by the experts in inflammasome research. It also contains two chemical compounds for priming and activation of the NLRP3 inflammasome. This economic starter set contains enough primary antibodies to perform at least 3 western blot experiments. |
THE STANDARD KO-extract validated Antibodies - Used and published by the Experts!
| PID | PRODUCT NAME | PROTOCOL |
| AG-20B-0042 | anti-Caspase-1 (p20) (mouse), mAb (Casper-1) | Measuring the Inflammasome (Download Protocol) Immunoblotting for Active Caspase-1 (Download Protocol) Inflammasome Activation and Inhibition in Primary Murine Bone Marrow-Derived Cells, and Assays for IL-1α, IL-1β, and Caspase-1 (Download Protocol) Assessing Caspase-1 Activation (Download Protocol) |
| AG-20B-0044 | anti-Caspase-1 (p10) (mouse), mAb (Casper-2) | Measuring the Inflammasome (Download Protocol) |
| AG-20B-0048 | anti-Caspase-1 (p20) (human), mAb (Bally-1) | Measuring the Inflammasome (Download Protocol) Cell-Free Assay for Inflammasome Activation (Download Protocol) |
| AG-20B-0014 |
anti-NLRP3/NALP3, mAb (Cryo-2) |
Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking (Download Protocol) Functional Reconstruction of NLRs in HEK293 Cells (Download Protocol) |
| AG-25B-0006 | anti-Asc, pAb (AL177) | Measuring Inflammasome Activation in Response to Bacterial Infection (Download Protocol) Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence (Download Protocol) Cell-Free Assay for Inflammasome Activation (Download Protocol) |
| AG-20B-0009 | anti-RIG-I, mAb (Alme-1) | The Human Papillomavirus E6 Oncoprotein Targets USP15 and TRIM25 To Suppress RIG-I-Mediated Innate Immune Signaling: C. Chiang, et al.; J. Virol. 92, e01737-17 (2018) [KO Validation] (Publication) |
| AG-20B-0010 | anti-ZBP1, mAb (Zippy-1) | Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1: H. Guo, et al.; Cell Death Dis. 9, 816 (2018) [KO Validation] (Publication) |
i) Cells or Tissues:
- Verify in the literature that primary cells, tissues or cell lines used express NLRP3 at detectable levels. Priming with LPS, PMA or other priming reagents (that activates NF-κB signaling) can be performed to increase NLRP3 levels.
- NLRP3 is expressed in many different cells or tissues, but in some cases (unexpected mutations in some cell subclones, others), levels of NLRP3 can be regulated leading to no or weaker signal in WB.
ii) SDS-PAGE gel:
- Percentage SDS-PAGE should be optimally <8%.
- All buffers (running, stacking, transfer) should be made fresh.
- Load at least 20-30µg per lane.
- Transfer is important since NLRP3 is a tricky protein not so easy to transfer.
- Transfer should be performed in a wet tank device overnight at 20V at 4°C .
- No transfer for 1h and also dry or semi-dry devices are NOT recommended.
iii) Primary antibody:
- Use freshly prepared antibody anti-NLRP3 (Cryo-2) at a dilution of 1/1’000. If protein is weakly expressed, lower dilution, such as 1/500 can be used.
iv) Secondary antibody:
- Use a freshly prepared anti-mouse IgG (AG-29B-0004E) antibody coupled to HRP (1/5’000) (or recommended dilution mentioned by the manufacturer if a different secondary anti-mouse IgG-HRP is used).
v) Positive controls:
- As positive control cell lines (that can be primed with ATP, PMA or LPS to increase NLRP3 levels), we recommend human monocyte-like cells, such as THP1 or U937 and mouse macrophage-like cells Raw264.7 or mouse monocyte /macrophage cell J774A.1.
Notes:
- Adipogen anti-NLRP3 antibody (Cryo-2) is one of the very few NLRP3 antibody validated using KO extracts. Many antibodies on the market are not validated and have been shown to be not specific.
- Adipogen anti-NLRP3 antibody (Cryo-2) detects the N-terminus Pyrin domain.
Co-culture protocol to study in vitro the role of inflammasomes in myeloid cells (macrophages) on T cells down regulation
A key observation of tissue injury, such as stroke and burn, is a state of systemic immunosuppression characterized by loss of T cells and the rise of infections. J. Zhu, et al. (2021) present an in vitro model for cell-cell interactions between innate (macrophages) and adaptive (T cells) immune cells. This protocol facilitates bone marrow-derived macrophages (BMDMs) and splenic T cells in a co-culture model. The procedure mimics injury-induced T cell death, which is driven by inflammasome activation in macrophages.
LIT: A macrophage-T cell coculture model for severe tissue injury-induced T cell death: J. Zhu, et al.; STAR Protocols 2, 100983 (2021) | Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade: S. Roth, et al.; Immunity 54, 648 (2021)
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