APAF1 Antibody [L11A23] | Primary Antibodies

Application: Reactivity: Mouse, Rat, Human

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:30

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
141 kDa, 125 kDa 140 kDa, 125 kDa
^*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 spleen tissue; Rat liver tissue; Human fetal kidney tissue; Human fetal heart tissue; A549 cells; HCT 116 cells; HAP1 cells; HEK-293T cells; HeLa cells; A20 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: 5%. 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.

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: 5%. 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
APAF1 Antibody [L11A23] detects endogenous levels of total APAF1 protein.

Subcellular Location
Cytoplasm

Uniprot ID
O14727

Clone
L11A23

Synonym(s)
KIAA0413; APAF1; Apoptotic protease-activating factor 1; APAF-1

Background
Apoptotic protease activating factor 1 (APAF1), the mammalian homolog of C. elegans CED-4, is a pivotal adaptor protein in the intrinsic apoptosis pathway, forming the apoptosome complex essential for caspase activation. APAF1 comprises an N-terminal CARD domain for caspase-9 recruitment, a central CED-4 homology domain (including nucleotide-binding and helical domains), and C-terminal WD40 repeats that regulate autoinhibition, with key residues like Arg265 and Asp244 facilitating dATP binding and hydrolysis to induce conformational activation. Its primary function involves binding cytosolic cytochrome c and dATP/ATP, oligomerizing into a wheel-like apoptosome that recruits and activates procaspase-9 via CARD-CARD interactions, thereby triggering the activation of downstream effector caspases, such as caspase-3, to execute apoptosis. This process is central to mitochondrial outer membrane permeabilization-mediated cell death, eliminating damaged cells in development, tissue homeostasis, and stress responses. Dysregulation links APAF1 to diseases: deficiency causes apoptosis resistance and hyperplasia, as in Apaf-1 knockout mice, while overexpression enhances p53-mediated neuronal apoptosis post-injury. APAF1 also emerges as a DNA sensor in innate immunity, with positively charged NB-ARC/WD40 surfaces binding cytosolic DNA before cytochrome c competition shifts it to apoptosome assembly.

Background

Apoptotic protease activating factor 1 (APAF1), the mammalian homolog of C. elegans CED-4, is a pivotal adaptor protein in the intrinsic apoptosis pathway, forming the apoptosome complex essential for caspase activation. APAF1 comprises an N-terminal CARD domain for caspase-9 recruitment, a central CED-4 homology domain (including nucleotide-binding and helical domains), and C-terminal WD40 repeats that regulate autoinhibition, with key residues like Arg265 and Asp244 facilitating dATP binding and hydrolysis to induce conformational activation. Its primary function involves binding cytosolic cytochrome c and dATP/ATP, oligomerizing into a wheel-like apoptosome that recruits and activates procaspase-9 via CARD-CARD interactions, thereby triggering the activation of downstream effector caspases, such as caspase-3, to execute apoptosis. This process is central to mitochondrial outer membrane permeabilization-mediated cell death, eliminating damaged cells in development, tissue homeostasis, and stress responses. Dysregulation links APAF1 to diseases: deficiency causes apoptosis resistance and hyperplasia, as in Apaf-1 knockout mice, while overexpression enhances p53-mediated neuronal apoptosis post-injury. APAF1 also emerges as a DNA sensor in innate immunity, with positively charged NB-ARC/WD40 surfaces binding cytosolic DNA before cytochrome c competition shifts it to apoptosome assembly.

References
https://pubmed.ncbi.nlm.nih.gov/26543158/ https://pubmed.ncbi.nlm.nih.gov/29765111/

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%