Datasheet

Biological Description

Specificity	Phospho-IKB α (Ser 36) Rabbit mAb recognizes endogenous levels of total IKBα protein when phosphorylated at Ser 36.
Background	Nuclear factor-kappa B (NF-κB) is a key family of transcription regulators within the NF-κB/Rel protein family, present in nearly all cell types. Its activity is tightly controlled by the inhibitor protein IκB, which binds to NF-κB dimers—either homologous or heterologous—forming a trimeric complex that keeps NF-κB sequestered and inactive in the cytoplasm. IκBα, the most prevalent member of the IκB family, has the ability to shuttle between the cytoplasm and nucleus, leading to a dynamic intracellular distribution. The human IκBα protein consists of 317 amino acids and has a molecular weight of 36 kDa. In its free form, IκBα exhibits a molten globule structure. Its N-terminal signal response domain (SRD) plays a crucial role in receiving phosphorylation, ubiquitination, and SUMOylation signals. This domain contains phosphorylation sites at Ser32, Ser36, and Tyr42, ubiquitination sites at Lys21 and Lys22, and SUMOylation sites at Lys21 and Lys38. The SRD is essential for activating NF-κB. The IκB kinase (IKK) complex, which can be triggered by lipopolysaccharides (LPS), viral proteins, reactive oxygen species, cytokines, and other stimuli, phosphorylates Ser32 and Ser36 in IκBα's SRD region. This phosphorylation leads to IκBα's ubiquitination and subsequent degradation by the proteasome, freeing NF-κB to enter the nucleus and activate target genes. Mutations in IκBα that result in gain-of-function changes at Ser32/Ser36 can lead to an autosomal dominant form of anhidrotic ectodermal dysplasia with immunodeficiency.

Specificity

Phospho-IKB α (Ser 36) Rabbit mAb recognizes endogenous levels of total IKBα protein when phosphorylated at Ser 36.

Background

Nuclear factor-kappa B (NF-κB) is a key family of transcription regulators within the NF-κB/Rel protein family, present in nearly all cell types. Its activity is tightly controlled by the inhibitor protein IκB, which binds to NF-κB dimers—either homologous or heterologous—forming a trimeric complex that keeps NF-κB sequestered and inactive in the cytoplasm. IκBα, the most prevalent member of the IκB family, has the ability to shuttle between the cytoplasm and nucleus, leading to a dynamic intracellular distribution. The human IκBα protein consists of 317 amino acids and has a molecular weight of 36 kDa. In its free form, IκBα exhibits a molten globule structure. Its N-terminal signal response domain (SRD) plays a crucial role in receiving phosphorylation, ubiquitination, and SUMOylation signals. This domain contains phosphorylation sites at Ser32, Ser36, and Tyr42, ubiquitination sites at Lys21 and Lys22, and SUMOylation sites at Lys21 and Lys38. The SRD is essential for activating NF-κB. The IκB kinase (IKK) complex, which can be triggered by lipopolysaccharides (LPS), viral proteins, reactive oxygen species, cytokines, and other stimuli, phosphorylates Ser32 and Ser36 in IκBα's SRD region. This phosphorylation leads to IκBα's ubiquitination and subsequent degradation by the proteasome, freeing NF-κB to enter the nucleus and activate target genes. Mutations in IκBα that result in gain-of-function changes at Ser32/Ser36 can lead to an autosomal dominant form of anhidrotic ectodermal dysplasia with immunodeficiency.

Usage Information

Application

WB, FCM

Dilution

WB	FCM
1:10000	1:100

Reactivity

Human, Mouse, Rat

Source

Rabbit

MW

35-40 kDa

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

Exprimental 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, Phosphatase 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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, 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 1:10000), 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 1126. 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.

Exprimental 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, Phosphatase 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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, 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 1:10000), 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

1126. 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.

References

[1] Wang X, et al. Front Cell Dev Biol. 2020 Nov 5;8:574706.

Application Data

WB

Validated by Selleck

Lane 1: C6
Lane 2: C6 (Calyculin A, 100ng/ml ,60 min)
Lane 3: C6 (Calyculin A, 100ng/ml ,60 min; Alkaline phosphatase-treated)