XAF1 Antibody [C23J4] | Primary Antibodies

Application: Reactivity: Human

Lane 1: Hela, Lane 2: Hela (IFN-α, 10 ng/ml, 16 h)

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 IP1:100

Application
WB, IP

Reactivity
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
32 kDa, 38 kDa

Positive Control	HT-29 cells (hIFN-α1, 10 ng/ml, overnight); HeLa cells (hIFN-α1, 10 ng/ml, overnight); MCF7 cells (hIFN-α1, 10 ng/ml, overnight); Jurkat cells (hIFN-α1, 10 ng/ml, overnight)
Negative Control	HT-29 cells; HeLa cells; MCF7 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. 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.

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.

Datasheet & SDS

Biological Description

Specificity
XAF1 Antibody [C23J4] detects endogenous levels of total XAF1 protein.

Subcellular Location
Cytoplasm, Mitochondrion, Nucleus

Uniprot ID
Q6GPH4

Clone
C23J4

Synonym(s)
XIAP-associated factor 1; XAF1

Background
X‑linked inhibitor of apoptosis‑associated factor 1 (XAF1) is a zinc finger–containing protein that functions as a pro‑apoptotic tumor suppressor by antagonizing the anti‑caspase activity of XIAP and modulating multiple stress‑signaling pathways. XAF1 contains several C2‑H2 zinc finger motifs that mediate protein–protein interactions, including direct binding to XIAP, p53, and components of the HIPK2–ZNF313 axis, allowing it to influence the stability and activity of these regulators rather than simply acting as a neutral adaptor. XAF1 is transcriptionally induced via STAT‑ and IRF‑1‑dependent circuits and functions as a feedback amplifier of interferon signaling, enhancing stress‑induced apoptosis and cellular sensitivity to exogenous death signals, while in p53‑proficient cells XAF1 forms a positive feedback loop with p53 by competing with the E3 ubiquitin ligase MDM2 for binding to p53, thereby stabilizing p53 and promoting HIPK2‑mediated p53 Ser46 phosphorylation and ZNF313‑dependent ubiquitination of p21, which collectively bias p53 outcomes toward apoptosis rather than cell‑cycle arrest. XAF1 engages non‑caspase‑dependent outputs, including autophagy induction via modulation of the Akt–p70S6K pathway and upregulation of Beclin‑1, and recent evidence indicates that XAF1 can be actively secreted from stressed tumor cells through an endolysosomal route to enhance T‑cell‑mediated tumor surveillance, linking intrinsic ER‑stress and DNA‑damage signaling to adaptive immune control. XAF1 expression is frequently suppressed by promoter hypermethylation or p53‑linked repression, and low XAF1 levels correlate with advanced stage, poor differentiation, and worse prognosis in multiple epithelial malignancies, whereas its restoration impairs tumor growth and sensitizes cells to cytotoxic agents.

Background

X‑linked inhibitor of apoptosis‑associated factor 1 (XAF1) is a zinc finger–containing protein that functions as a pro‑apoptotic tumor suppressor by antagonizing the anti‑caspase activity of XIAP and modulating multiple stress‑signaling pathways. XAF1 contains several C2‑H2 zinc finger motifs that mediate protein–protein interactions, including direct binding to XIAP, p53, and components of the HIPK2–ZNF313 axis, allowing it to influence the stability and activity of these regulators rather than simply acting as a neutral adaptor. XAF1 is transcriptionally induced via STAT‑ and IRF‑1‑dependent circuits and functions as a feedback amplifier of interferon signaling, enhancing stress‑induced apoptosis and cellular sensitivity to exogenous death signals, while in p53‑proficient cells XAF1 forms a positive feedback loop with p53 by competing with the E3 ubiquitin ligase MDM2 for binding to p53, thereby stabilizing p53 and promoting HIPK2‑mediated p53 Ser46 phosphorylation and ZNF313‑dependent ubiquitination of p21, which collectively bias p53 outcomes toward apoptosis rather than cell‑cycle arrest. XAF1 engages non‑caspase‑dependent outputs, including autophagy induction via modulation of the Akt–p70S6K pathway and upregulation of Beclin‑1, and recent evidence indicates that XAF1 can be actively secreted from stressed tumor cells through an endolysosomal route to enhance T‑cell‑mediated tumor surveillance, linking intrinsic ER‑stress and DNA‑damage signaling to adaptive immune control. XAF1 expression is frequently suppressed by promoter hypermethylation or p53‑linked repression, and low XAF1 levels correlate with advanced stage, poor differentiation, and worse prognosis in multiple epithelial malignancies, whereas its restoration impairs tumor growth and sensitizes cells to cytotoxic agents.

References
https://pubmed.ncbi.nlm.nih.gov/25313037/ https://pubmed.ncbi.nlm.nih.gov/30042418/

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%