The Pyroptosis Antibody Sampler Kit provides an economical means of detecting proteins that are used as readouts for pyroptosis. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.
Specificity / Sensitivity
Gasdermin D (E8G3F) Rabbit mAb recognizes endogenous levels of total Gasdermin D protein. This antibody recognizes the 30 kDa amino terminal fragment produced during pyroptosis by caspase-1, a 43 kDa fragment produced by caspase-3, as well as a 21 kDa fragment produced by cleavage at both sites. Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb detects the N-terminal fragment of human Gasdermin D protein only when cleaved at Asp275. Caspase-1 (D7F10) Rabbit mAb detects endogenous levels of full length human caspase-1 and the activated p20 subunit by overexpression. Cleaved Caspase-1 (Asp297) (D57A2) Rabbit mAb detects the p20 subunit of human caspase-1 upon cleavage at Asp275. IL-1β (D3U3E) Rabbit mAb can recognize endogenous levels of full-length protein, but does not detect endogenous levels of the mature IL-1β. It can detect up to 100 pg of recombinant mature IL-1β. Cleaved IL-1β (Asp116) (D3A3Z) Rabbit mAb detects endogenous levels of mature IL-1β protein when cleaved at Asp116. Caspase-4 Antibody detects endogenous levels of total caspase-4 and intermediate forms at 40 and 32 kDa. Caspase-5 (D3G4W) Rabbit mAb detects endogenous levels of caspase-5. HMGB1 (D3E5) Rabbit mAb detects endogenous levels of total HMGB1. It does not cross-react with other HMGB proteins, including HMGB2 and HMGB3.
Source / Purification
Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Trp130 of human Gasdermin D, residues within the p20 subunit of caspase-1, Asp297 of human caspase-1, Asp116 of human IL-1β, Pro154 of human caspase-5, and Ala137 of human HMGB1. IL-1β (D3U3E) Rabbit mAb is produced by immunizing animals with a recombinant IL-1β protein. Polyclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile125 of human caspase-4. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.
Pyroptosis is a regulated pathway of cell death with morphological features of necrosis, including cell swelling, plasma membrane pore formation, and engagement of an inflammatory response with the release of a number of damage-associated molecular patterns (DAMPs), such as HMGB1 and inflammatory cytokines like IL-1β and IL-18 (1,2). Pyroptosis is generally induced in cells of the innate immune system, such as monocytes, macrophages, and dendritic cells in the presence of pathogen-associated molecular patterns (PAMPs) expressed on microbial pathogens or by cell-derived DAMPs. It is induced through assembly of inflammasomes triggering proteolytic activation of caspase-1 which then cleaves inflammatory cytokines like IL-1β and IL-18 to their mature forms (3). A critical feature of pyroptosis is the cleavage of Gasdermin D by caspase-1 and mouse caspase-11 (or human caspase-4/5) (4-6). Upon cleavage, the N-terminal fragment of Gasdermin D oligomerizes to form a pore, allowing secretion of inflammatory DAMPs and cytokines. Canonical inflammasome assembly typically consists of a cytosolic-pattern recognition receptor (PPR; a nucleotide binding domain and leucine-rich repeat [NLR] or AIM2-like family members), an adaptor protein (ASC/TMS1), and pro-caspase-1. Distinct inflammasome complexes can recognize distinct PAMPs and DAMPs to trigger pyroptosis. The best characterized pathway triggered by the NLR, NLRP3, occurs through a two-step process. The first step is a priming signal, NF-κB is activated to induce the expression of a number of inflammasome components including NLRP3, pro-IL-1β, and pro-IL-18. In the second activation step, caspase-1 is activated and Gasdermin D and cytokines are proteolytically activated. In a non-canonical pathway, caspase-4 and caspase-5 can directly trigger Gasdermin D cleavage in monocytes following LPS stimulation (5,7).