IKB alpha Recombinant Rabbit Monoclonal Antibody [SZ00-07]

Overview

Product Name

IKB alpha Recombinant Rabbit Monoclonal Antibody [SZ00-07] - BSA and Azide free

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within human IKB alpha aa 1-50.

Species Reactivity

Human, Mouse, Rat (Predicted: Zebrafish)

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, IP, FC

Target Molecular Weight

Predicted band size: 36 kDa

Positive Control

HeLa cell lysate, SK-Br-3 cell lysate, NIH/3T3 cell lysate, RAW264.7 cell lysate, PC-12 cell lysate, Hela, MCF-7, SHG-44, human kidney tissue, mouse kidney tissue, mouse brain tissue, mouse stomach tissue.

Conjugation

unconjugated

Clone Number

SZ00-07

Reactivity Data

Tested

Verified (internally validated) Published

Reported in literature (not internally validated) Predicted

Predicted reactive (based on sequence homology) Not recommended

Not recommended (failed internal validation)

	WB	IF-Cell	IF-Tissue	IHC-P	IP	FC
Human
Mouse
Rat
Zebrafish

Product Features

Form

Liquid

Concentration

1mg/ml

Storage Instructions

Store at 2-8℃. Avoid freeze.

Storage Buffer

1*PBS (pH7.4).

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

Target

Function

NFKB1 or NFKB2 is bound to REL, RELA, or RELB to form the NFKB complex. The NFKB complex is inhibited by I-kappa-B proteins, which inactivate NF-kappa-B by trapping it in the cytoplasm. Phosphorylation of serine residues on the I-kappa-B proteins by kinases marks them for destruction via the ubiquitination pathway, thereby allowing activation of the NF-kappa-B complex. Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime.

Background References

1. Bai X et al. Prostaglandin E2 stimulates 1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF- B pathway. Sci Rep 4:6538 (2014).

2. Broderick TL et al. Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart. ISRN Endocrinol 2012:736860 (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

SwissProt: Q63746 Rat

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

This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Western blot analysis of IKB alpha on different lysates with Rabbit anti-IKB alpha antibody (ET1603-6) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa cell lysate
Lane 3: SK-Br-3 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: RAW264.7 cell lysate
Lane 6: PC-12 cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 1 minute;

12% 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 (ET1603-6) at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
IKB alpha was immunoprecipitated from 0.5 mg Hela whole cell lysates with ET1603-6 at 2 μg/mL. Western blot was performed from the immunoprecipitate using ET1603-6 at 1/5,000 dilution for 45 minutes at room temperature. Goat anti-Rabbit IgG-HRP Secondary Antibody (HA1001) was used at 1:300,000 dilution for 30 minutes at room temperature.

Lane 1: Hela whole cell lysates at 10 μg;
Lane 2: IKB alpha (ET1603-6) IP in Hela whole cell lysates;
Lane 3: Rabbit IgG instead of IKB alpha (ET1603-6) in Hela whole cell lysates.

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

Exposure time: 1 minute;

12% SDS-PAGE gel.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-IKB alpha antibody (ET1603-6) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-6) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-IKB alpha antibody (ET1603-6) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-6) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-IKB alpha antibody (ET1603-6) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-6) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Application: IF-Tissue

Species: Human

Site: kidney

Sample: Paraffin-embedded section

Antibody concentration: 1/1,000
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of HeLa cells labeling IKB alpha with Rabbit anti-IKB alpha antibody (ET1603-6) 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-IKB alpha antibody (ET1603-6) 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.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of NIH/3T3 cells labeling IKB alpha with Rabbit anti-IKB alpha antibody (ET1603-6) 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-IKB alpha antibody (ET1603-6) 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.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of PC-12 cells labeling IKB alpha with Rabbit anti-IKB alpha antibody (ET1603-6) 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-IKB alpha antibody (ET1603-6) 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.
This data was developed using ET1603-6, the same antibody clone in a different buffer formulation.
Flow cytometric analysis of HeLa cells labeling IKB alpha.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1603-6, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).