pro Caspase-1+p10+p12 Antibody [H3L1]

Application: Reactivity: Mouse, Rat, Human

Lane 1: THP-1 (LPS, 0.1 µg/mL, 3 h; ATP, 5 mM, 15 min)

Experiment Essentials

The transcriptional products of the procaspase-1 gene undergo alternative splicing, thereby generating a variety of distinct isoforms. These protein isoforms differ in molecular weight, so multiple specific bands can be observed in the range of 30~45 kDa during protein electrophoresis. The 50 kDa band corresponds to caspase-1 α, the most widely expressed isoform.

Usage Information

Dilution
WB1:1000 IP1:100

Application
WB, IP

Reactivity
Mouse, Rat, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
45 kDa 10 kDa, 30-45kDa, 50kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Rat lung tissue; Human fetal lung tissue; Rat spleen tissue; Mouse spleen tissue; Mouse brain tissue; Rat brain tissue; RAW 264.7 cells; THP-1 cells; J774A.1 cells
Negative Control	NCI-H1299 cells; A549 cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
pro Caspase-1+p10+p12 Antibody (Rabbit mAb) [H3L1] detects endogenous levels of total pro Caspase-1, p10, p12 protein.

Subcellular Location
Cell membrane, Cytoplasm, Membrane

Uniprot ID
P29466

Clone
H3L1

Synonym(s)
IL1BC, IL1BCE, CASP1, Caspase-1, CASP-1, Interleukin-1 beta convertase, Interleukin-1 beta-converting enzyme, p45, IL-1BC, ICE, IL-1 beta-converting enzyme

Background
Pro caspase‑1 is the inactive zymogen of the inflammatory caspase‑1 that contains an N‑terminal caspase recruitment domain (CARD) followed by a large catalytic subunit (p20) and a small subunit (p10/p12 region), and the term “pro caspase‑1 + p10 + p12” refers to detection of both the full‑length precursor and its processed small subunits that together define the activation status of caspase‑1 within inflammasome pathways. The zymogen resides in the cytosol of myeloid and other cells and is recruited via CARD–CARD interactions to inflammasome platforms built by pattern‑recognition receptors such as NLRP3, NLRC4, AIM2, or other NLRs and adaptor ASC, which bring multiple pro caspase‑1 molecules into proximity and promote autoproteolytic processing at defined internal aspartate residues to generate p20 and p10/p12 fragments that assemble into an active heterotetramer. The active enzyme cleaves the pro‑forms of IL‑1β and IL‑18 to their mature secreted cytokines and also processes gasdermin D to release its N‑terminal pore‑forming domain, so caspase‑1 activation via its p20/p10 complex couples pathogen‑ or damage‑associated molecular pattern sensing to secretion of key pro‑inflammatory cytokines and to pyroptotic lytic cell death that facilitates pathogen clearance and inflammatory signaling. Beyond canonical inflammasomes, caspase‑1 participates in alternative activation routes, interacts with RIP2 and NF‑κB signaling components, and can influence glycolytic enzymes, caspase‑7 activation, and unconventional protein secretion, indicating broader roles in metabolic adaptation, cell survival, and non‑classical secretion in a context‑dependent manner. The balance between the unprocessed pro form and the p10/p12‑containing active subunits therefore provides a direct biochemical readout of inflammasome engagement and caspase‑1 activation state in tissues and experimental systems, and antibodies that recognize epitopes present in pro caspase‑1 and in the p10/p12 region allow detection of both pools to monitor zymogen availability and activation‑associated cleavage within a single assay. Dysregulated caspase‑1 activity contributes to autoinflammatory and inflammatory diseases, including cryopyrin‑associated periodic syndromes, gout, rheumatoid arthritis, inflammatory bowel disease, obesity‑associated metabolic disorders, cardiovascular disease, and some cancers, through excessive IL‑1β/IL‑18 production and pyroptosis, while insufficient activation impairs host defense against bacterial and viral infections.

Background

Pro caspase‑1 is the inactive zymogen of the inflammatory caspase‑1 that contains an N‑terminal caspase recruitment domain (CARD) followed by a large catalytic subunit (p20) and a small subunit (p10/p12 region), and the term “pro caspase‑1 + p10 + p12” refers to detection of both the full‑length precursor and its processed small subunits that together define the activation status of caspase‑1 within inflammasome pathways. The zymogen resides in the cytosol of myeloid and other cells and is recruited via CARD–CARD interactions to inflammasome platforms built by pattern‑recognition receptors such as NLRP3, NLRC4, AIM2, or other NLRs and adaptor ASC, which bring multiple pro caspase‑1 molecules into proximity and promote autoproteolytic processing at defined internal aspartate residues to generate p20 and p10/p12 fragments that assemble into an active heterotetramer. The active enzyme cleaves the pro‑forms of IL‑1β and IL‑18 to their mature secreted cytokines and also processes gasdermin D to release its N‑terminal pore‑forming domain, so caspase‑1 activation via its p20/p10 complex couples pathogen‑ or damage‑associated molecular pattern sensing to secretion of key pro‑inflammatory cytokines and to pyroptotic lytic cell death that facilitates pathogen clearance and inflammatory signaling. Beyond canonical inflammasomes, caspase‑1 participates in alternative activation routes, interacts with RIP2 and NF‑κB signaling components, and can influence glycolytic enzymes, caspase‑7 activation, and unconventional protein secretion, indicating broader roles in metabolic adaptation, cell survival, and non‑classical secretion in a context‑dependent manner. The balance between the unprocessed pro form and the p10/p12‑containing active subunits therefore provides a direct biochemical readout of inflammasome engagement and caspase‑1 activation state in tissues and experimental systems, and antibodies that recognize epitopes present in pro caspase‑1 and in the p10/p12 region allow detection of both pools to monitor zymogen availability and activation‑associated cleavage within a single assay. Dysregulated caspase‑1 activity contributes to autoinflammatory and inflammatory diseases, including cryopyrin‑associated periodic syndromes, gout, rheumatoid arthritis, inflammatory bowel disease, obesity‑associated metabolic disorders, cardiovascular disease, and some cancers, through excessive IL‑1β/IL‑18 production and pyroptosis, while insufficient activation impairs host defense against bacterial and viral infections.

References
https://pubmed.ncbi.nlm.nih.gov/27450556/ https://pubmed.ncbi.nlm.nih.gov/26059719/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

pro Caspase-1+p10+p12 Antibody (Rabbit mAb) [H3L1]