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Phospho-IKB alpha (S32) Recombinant Rabbit Monoclonal Antibody [ST53-05]

Usd: 385

Specification

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Catalog# ET1609-78

Phospho-IKB alpha (S32) Recombinant Rabbit Monoclonal Antibody [ST53-05]

Application

  • WB

  • IF-Cell

  • IP

Reactivity

  • Human

  • Mouse

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Phospho-IKB alpha (S32) Recombinant Rabbit Monoclonal Antibody [ST53-05]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser32 of human IKB alpha.

Species Reactivity

Human, Mouse

Validated Applications

WB, IF-Cell, IP

Molecular Weight

Predicted band size: 36 kDa

Positive Control

HeLa treated with 20ng/mL TNF-α for 5 minutes cell lysate, HeLa treated with 100nM Calyculin A for 30 minutes cell lysate, RAW264.7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate, NIH/3T3 cells were starved for 18 hours then treated with 20 ng/ml TNF alpha for 5 minutes, HeLa.

Conjugation

unconjugated

Clone Number

ST53-05

RRID

AB_3069887

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000-1:5,000

  • IF-Cell

  • 1:50-1:200

  • IP

  • Use at an assay dependent concentration.

Target

Function

On the basis of both functional and structural considerations, members of the IκB family of proteins can be divided into four groups. The first of these groups, IκB-α, includes the avian protein pp40 and the mammalian MAD-3, both of which inhibit binding of p50-p65 NFkB complex or Rel protein to their cognate binding sites but do not inhibit the binding of p50 homodimer to κB sites, suggesting that the IκB-α family binds to the p65 subunit of p50-p65 heterocomplex through ankyrin repeats. The second member of the IκB family is represented by a protein designated IκB-β. The third group of IκB proteins is represented by IκB-γ, which is identical in sequence with the C-terminal domain of the p110 precursor of NFkB p50 and is expressed predominantly in lymphoid cells. An additional IκB family member, IκB-ε, has several phosphorylated forms and is primarily found complexed with Rel A and/or c-Rel.

Background References

1. Liu Y et al. The natural compound magnolol inhibits invasion and exhibits potential in human breast cancer therapy. Sci Rep 3:3098 (2013).

2. Kiefel H et al. EMT-associated up-regulation of L1CAM provides insights into L1CAM-mediated integrin signalling and NF- B activation. Carcinogenesis 33:1919-29 (2012).

Sequence Similarity

Belongs to the NF-kappa-B inhibitor family.

Post-translational Modification

Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. Phosphorylation at positions 32 and 36 is prerequisite to recognition by UBE2D3 leading to polyubiquitination and subsequent degradation.; Sumoylated; sumoylation requires the presence of the nuclear import signal. Sumoylation blocks ubiquitination and proteasome-mediated degradation of the protein thereby increasing the protein stability.; Monoubiquitinated at Lys-21 and/or Lys-22 by UBE2D3. Ubiquitin chain elongation is then performed by CDC34 in cooperation with the SCF(FBXW11) E3 ligase complex, building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin. The resulting polyubiquitination leads to protein degradation. Also ubiquitinated by SCF(BTRC) following stimulus-dependent phosphorylation at Ser-32 and Ser-36.; Deubiquitinated by porcine reproductive and respiratory syndrome virus Nsp2 protein, which thereby interferes with NFKBIA degradation and impairs subsequent NF-kappa-B activation.

Subcellular Location

Cytoplasm, Nucleus.

UNIPROT

SwissProt: P25963 Human

SwissProt: Q9Z1E3 Mouse

Synonyms

I kappa B alpha antibody

I-kappa-B-alpha antibody

IkappaBalpha antibody

IkB-alpha antibody

IKBA antibody

IKBA_HUMAN antibody

IKBalpha antibody

MAD 3 antibody

MAD3 antibody

Major histocompatibility complex enhancer-binding protein MAD3 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-IKB alpha (S32) on different lysates with Rabbit anti-Phospho-IKB alpha (S32) antibody (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: HeLa treated with 20ng/mL TNF-α for 5 minutes cell lysate<br />Lane 3: HeLa treated with 20ng/mL TNF-α for 5 minutes cell lysate, then the membrane treated with λpp for 1 hour<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 36 kDa<br />Observed band size: 36 kDa<br /><br />Exposure time: 1 minute 2 seconds;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-IKB alpha (S32) on different lysates with Rabbit anti-Phospho-IKB alpha (S32) antibody (ET1609-78) at 1/1,000 dilution.

    Lane 1: HeLa cell lysate
    Lane 2: HeLa treated with 20ng/mL TNF-α for 5 minutes cell lysate
    Lane 3: HeLa treated with 20ng/mL TNF-α for 5 minutes cell lysate, then the membrane treated with λpp for 1 hour

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

    Exposure time: 1 minute 2 seconds;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-78) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-IKB alpha (S32) on different lysates with Rabbit anti-Phospho-IKB alpha (S32) antibody (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>) at 1/5,000 dilution.<br /><br />Lane 1: HeLa cell lysate (20 µg/Lane)<br />Lane 2: HeLa treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)<br />Lane 3: RAW264.7 cell lysate (20 µg/Lane)<br />Lane 4: RAW264.7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate (20 µg/Lane)<br /><br />Predicted band size: 36 kDa<br />Observed band size: 36 kDa<br /><br />Exposure time: 59 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>) at 1/5,000 dilution was used in primary antibody dilution (<a href="/products/K1803" style="font-weight: bold;text-decoration: underline;">K1803</a>) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-IKB alpha (S32) on different lysates with Rabbit anti-Phospho-IKB alpha (S32) antibody (ET1609-78) at 1/5,000 dilution.

    Lane 1: HeLa cell lysate (20 µg/Lane)
    Lane 2: HeLa treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)
    Lane 3: RAW264.7 cell lysate (20 µg/Lane)
    Lane 4: RAW264.7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate (20 µg/Lane)

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

    Exposure time: 59 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-78) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-IKB alpha (S32) on NIH-3T3 cell lysates.<br /><br />Lane 1: NIH/3T3 cells were starved for 18 hours, whole cell lysate, 10 ug/lane.<br />Lane 2: NIH/3T3 cells were starved for 18 hours, then treated with 20 ng/ml TNF alpha for 5 minutes, whole cell lysates, 10 ug/lane.<br />Lane 3: NIH/3T3 cells were starved for 18 hours, then treated with 20 ng/ml TNF alpha for 10 minutes, whole cell lysates, 10 ug/lane.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-IKB alpha (S32) (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>, 1/1,000) , Anti-IKB alpha antibody ( <a href="/products/ET1603-6" style="font-weight: bold;text-decoration: underline;">ET1603-6</a>, 1/2,000) and Anti-GAPDH antibody (<a href="/products/ET1601-4" style="font-weight: bold;text-decoration: underline;">ET1601-4</a>, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1:200,000 dilution was used for 1 hour at room temperature.<br /><br />Predicted band size: 36 kDa<br />Observed band size: 36 kDa<br /><br />Exposure time: 1 minute

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-IKB alpha (S32) on NIH-3T3 cell lysates.

    Lane 1: NIH/3T3 cells were starved for 18 hours, whole cell lysate, 10 ug/lane.
    Lane 2: NIH/3T3 cells were starved for 18 hours, then treated with 20 ng/ml TNF alpha for 5 minutes, whole cell lysates, 10 ug/lane.
    Lane 3: NIH/3T3 cells were starved for 18 hours, then treated with 20 ng/ml TNF alpha for 10 minutes, whole cell lysates, 10 ug/lane.

    Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-IKB alpha (S32) (ET1609-78, 1/1,000) , Anti-IKB alpha antibody ( ET1603-6, 1/2,000) and Anti-GAPDH antibody (ET1601-4, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

    Exposure time: 1 minute

  • Immunocytochemistry analysis of HeLa cells labeling Phospho-IKB alpha (S32) with Rabbit anti-Phospho-IKB alpha (S32) antibody (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-IKB alpha (S32) antibody (<a href="/products/ET1609-78" style="font-weight: bold;text-decoration: underline;">ET1609-78</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of HeLa cells labeling Phospho-IKB alpha (S32) with Rabbit anti-Phospho-IKB alpha (S32) antibody (ET1609-78) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-IKB alpha (S32) antibody (ET1609-78) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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