Caspase9 Antibody [A3K12] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat, Hamster, Monkey

Lane 1: Jurkat, Lane 2: Jurkat (staurosporine treated)

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.
Exposure time of at least 120s is recommended.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat, Hamster, Monkey

Source
Mouse 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
35-51 kDa

Positive Control	Jurkat cells; L929 cells; C6 cells
Negative Control

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. 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) and put the sample on ice for 5 min. 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) and put the sample on ice for 5 min. 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, 60 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. (Exposure time of at least 120s is recommended)

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.

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) and put the sample on ice for 5 min.

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) and put the sample on ice for 5 min.

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, 60 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. (Exposure time of at least 120s is recommended)

Datasheet & SDS

Biological Description

Specificity
Caspase9 Antibody [A3K12] detects endogenous levels of total Caspase9 protein.

Uniprot ID
P55211

Clone
A3K12

Synonym(s)
Caspase-9; CASP-9; Apoptotic protease Mch-6; Apoptotic protease-activating factor 3 (APAF-3); ICE-like apoptotic protease 6 (ICE-LAP6); CASP9; MCH6

Background
Caspase-9 is an initiator caspase central to the intrinsic apoptosis pathway, acting at the apex of the mitochondrial death signaling cascade. Upon cytochrome c release from mitochondria, caspase-9 assembles into the Apaf-1 apoptosome. This process involves induced proximity oligomerization via Apaf-1 CARD domain interactions, leading to autocatalytic cleavage at Asp315 and formation of an active p35/p12 heterodimer. A secondary cleavage at Asp330 yields a p37 fragment, further amplifying effector caspase activation. The apoptosome-bound caspase-9 demonstrates over 100-fold enhanced activity due to allosteric stabilization of its active conformation, maintained by Apaf-1 even without continued dATP presence after assembly. This holoenzyme efficiently activates caspase-3 and caspase-7, initiating the executioner phase, which results in PARP cleavage, DNA fragmentation, and cytoskeletal collapse. Physiological inhibition occurs through Xiap, which binds the active site and BIR2 domain of processed caspase-9; however, caspase-3 feedback cleavage of Xiap relieves this inhibition, ensuring irreversible apoptosis commitment. Alternative splicing produces the dominant-negative caspase-9S isoform, which lacks catalytic activity and competes for apoptosome docking. Caspase-9 is essential for developmental sculpting, DNA damage surveillance, and stress-induced clearance in neurons and lymphocytes. It serves as the canonical marker for intrinsic apoptosis in research assays using fluorogenic substrates or dominant-negative mutants in flow cytometry. Caspase-9 deficiency leads to severe lymphoproliferation and autoimmunity, while APAF-1 methylation in cancer silences the pathway.

Background

Caspase-9 is an initiator caspase central to the intrinsic apoptosis pathway, acting at the apex of the mitochondrial death signaling cascade. Upon cytochrome c release from mitochondria, caspase-9 assembles into the Apaf-1 apoptosome. This process involves induced proximity oligomerization via Apaf-1 CARD domain interactions, leading to autocatalytic cleavage at Asp315 and formation of an active p35/p12 heterodimer. A secondary cleavage at Asp330 yields a p37 fragment, further amplifying effector caspase activation. The apoptosome-bound caspase-9 demonstrates over 100-fold enhanced activity due to allosteric stabilization of its active conformation, maintained by Apaf-1 even without continued dATP presence after assembly. This holoenzyme efficiently activates caspase-3 and caspase-7, initiating the executioner phase, which results in PARP cleavage, DNA fragmentation, and cytoskeletal collapse. Physiological inhibition occurs through Xiap, which binds the active site and BIR2 domain of processed caspase-9; however, caspase-3 feedback cleavage of Xiap relieves this inhibition, ensuring irreversible apoptosis commitment. Alternative splicing produces the dominant-negative caspase-9S isoform, which lacks catalytic activity and competes for apoptosome docking. Caspase-9 is essential for developmental sculpting, DNA damage surveillance, and stress-induced clearance in neurons and lymphocytes. It serves as the canonical marker for intrinsic apoptosis in research assays using fluorogenic substrates or dominant-negative mutants in flow cytometry. Caspase-9 deficiency leads to severe lymphoproliferation and autoimmunity, while APAF-1 methylation in cancer silences the pathway.

References
https://pubmed.ncbi.nlm.nih.gov/10617566/ https://pubmed.ncbi.nlm.nih.gov/11904389/

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%