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Anti-Phospho-IκBα (Ser32/36) Mouse Antibody [F24N7]

Catalog No.: F0197

Application: Reactivity: Human, Mouse, Rat, Monkey

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.
Exposure time of at least 150s is recommended.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat, Monkey

Source
Mouse

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
40 kDa

Positive Control	NIH-3T3 cell (treated with TNF-α (20 ng/ml) for 5 mins); HeLa cell (IFN-α treated (20 ng/ml) for 5 mins); NIH-3T3 cell ( treated with INF-α (20 ng/ml) for 5 mins); THP-1 cell (differentiated with TPA ( 80 nM for 24 h) and treated with 1 μg/ml LPS for 5-4h); HeLa cell (treated with human TNF⍺ (20ng/mL; 5min)
Negative Control

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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 ( recommending 5% BSA 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 150s is recommended)

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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 ( recommending 5% BSA 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 150s is recommended)

Datasheet & SDS

Biological Description

Specificity
Phospho-IκBα (Ser32/36) Mouse mAb detects endogenous levels of IκBα only when phosphorylated at Ser32/36.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
P25963

Clone
F24N7

Synonym(s)
NF-kappa-B inhibitor alpha, I-kappa-B-alpha (IkB-alpha; IkappaBalpha), Major histocompatibility complex enhancer-binding protein MAD3, NFKBIA, IKBA, MAD3, NFKBI

Background
The IκB kinase (IKK) complex serves as a central signaling hub for the activation of the NF-κB pathway. This complex is composed of two catalytic subunits, IKKα and IKKβ, which are serine/threonine kinases, and a regulatory subunit known as NEMO (NF-κB essential modulator), also referred to as IKKγ. The IKK complex integrates a wide range of upstream signals that activate NF-κB, leading to the phosphorylation of IκB proteins, NF-κB subunits, and other cellular targets. In unstimulated cells, NF-κB transcription factors are maintained in an inactive state in the cytoplasm by binding to inhibitory IκB proteins, such as IκBα, IκBβ, and IκBε, or to the precursor proteins p100 and p105. These inhibitory proteins prevent NF-κB from entering the nucleus and initiating gene transcription. Upon stimulation, IκB proteins are phosphorylated at specific serine residues—particularly Ser32 and Ser36 in the case of IκBα—by the activated IKK complex. This phosphorylation marks IκB for recognition by the SCFβTrCP E3 ubiquitin ligase complex, leading to its ubiquitination and subsequent degradation via the proteasome. The degradation of IκB releases NF-κB dimers, which then translocate to the nucleus where they drive the transcription of target genes. A wide variety of external signals, including inflammatory cytokines, growth factors, and chemokines, can trigger this cascade, ultimately resulting in the phosphorylation of IκBα and activation of NF-κB-dependent gene expression.

Background

The IκB kinase (IKK) complex serves as a central signaling hub for the activation of the NF-κB pathway. This complex is composed of two catalytic subunits, IKKα and IKKβ, which are serine/threonine kinases, and a regulatory subunit known as NEMO (NF-κB essential modulator), also referred to as IKKγ. The IKK complex integrates a wide range of upstream signals that activate NF-κB, leading to the phosphorylation of IκB proteins, NF-κB subunits, and other cellular targets. In unstimulated cells, NF-κB transcription factors are maintained in an inactive state in the cytoplasm by binding to inhibitory IκB proteins, such as IκBα, IκBβ, and IκBε, or to the precursor proteins p100 and p105. These inhibitory proteins prevent NF-κB from entering the nucleus and initiating gene transcription. Upon stimulation, IκB proteins are phosphorylated at specific serine residues—particularly Ser32 and Ser36 in the case of IκBα—by the activated IKK complex. This phosphorylation marks IκB for recognition by the SCFβTrCP E3 ubiquitin ligase complex, leading to its ubiquitination and subsequent degradation via the proteasome. The degradation of IκB releases NF-κB dimers, which then translocate to the nucleus where they drive the transcription of target genes. A wide variety of external signals, including inflammatory cytokines, growth factors, and chemokines, can trigger this cascade, ultimately resulting in the phosphorylation of IκBα and activation of NF-κB-dependent gene expression.

References
https://pubmed.ncbi.nlm.nih.gov/24375677/ https://pubmed.ncbi.nlm.nih.gov/7878466/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

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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%