Rabbit Anti- phospho-IKB alpha (Tyr305) antibody

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IKB alpha (phospho Y305); p-IKB alpha (phospho Y305); NFKBIA(phospho Y305); Inhibitor of KB alpha; I kappa B alpha; I(Kappa)B(alpha); IkappaBalpha; IKBA; IKBalpha; IkB-alpha; MAD 3; MAD3; Major histocompatibility complex enhancer binding protein MAD3; NF

Cat:

SL5515R

Species Reactivity：

Human,Mouse,Rat,(predicted: Pig,Cow,Rabbit,Sheep,)

Immunogen：

KLH conjugated Synthesised phosphopeptide derived from human IKB alpha around the phosphorylation site of Tyr305:LP(p-Y)DD

Format：

Liquid

Storage instructions：

Shipped at 4℃. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles.

Concentration：

1mg/ml

Clonality：

Polyclonal

Isotype：

IgG

Applications：

WB=1:500-2000ELISA=1:5000-10000IHC-P=1:100-500IHC-F=1:100-500Flow-Cyt=1μg/TestIF=1:100-500（Paraffin sections need to do antigen repair）not yet tested in other applications.optimal dilutions/concentrations should be determined by the end user.

Host：

Rabbit

Product Overview：

Sample:293T Cell (Human) Lysate at 40 ugPrimary: Anti-p-NFKBIA(Tyr305)(SL5515R)at 1/300 dilutionSecondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilutionPredicted band size: 35 kDObserved band size: 38 kDParaformaldehyde-fixed, paraffin embedded (rat stomach); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat lymphoid); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat stomach); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat adrenal gland); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305) ) Polyclonal Antibody, Unconjugated (SL5515R) at 1:400 overnight at 4°C, followed by a conjugated secondary antibody (sp-0023) for 20 minutes and DAB staining.Tissue/cell: human colon carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min; Incubation: Anti-NFKBIA Polyclonal Antibody, Unconjugated(SL5515R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) stainingParaformaldehyde-fixed, paraffin embedded (Mouse liver); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (p-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Tissue/cell:Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,SLC0005) at 37°C for 20 min; Antibody incubation with (phospho-IKB alpha (Tyr305)) polyclonal Antibody, Unconjugated (SL5515R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.Blank control: Hela. Primary Antibody (green line): Rabbit Anti- phospho-IKB alpha (Tyr305) antibody (SL5515R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-AF647Dilution: 1μg /test. ProtocolThe cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.Blank control (blue line): Hela (fixed with 70% methanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃). Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (SL5515R),Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE,Dilution: 1μg /test. Blank control (blue line): Jurkat (fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice). Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (SL5515R),Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC,Dilution: 1μg /test.

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Unit:

Price: $256.00

Product PDFs

Datasheet: Down Datasheet

Other Information
Citations
Protein Attributes
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This gene encodes a member of the NF-kappa-B inhibitor family, which contain multiple ankrin repeat domains. The encoded protein interacts with REL dimers to inhibit NF-kappa-B/REL complexes which are involved in inflammatory responses. The encoded protein moves between the cytoplasm and the nucleus via a nuclear localization signal and CRM1-mediated nuclear export. Mutations in this gene have been found in ectodermal dysplasia anhidrotic with T-cell immunodeficiency autosomal dominant disease. [provided by RefSeq, Aug 2011].

Function:
Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.

Subunit:
Interacts with RELA; the interaction requires the nuclear import signal. Interacts with NKIRAS1 and NKIRAS2. Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, IKBKAP and MAP3K14. Interacts with HBV protein X. Interacts with RWDD3; the interaction enhances sumoylation. Interacts (when phosphorylated at the 2 serine residues in the destruction motif D-S-G-X(2,3,4)-S) with BTRC. Associates with the SCF(BTRC) complex, composed of SKP1, CUL1 and BTRC; the association is mediated via interaction with BTRC. Part of a SCF(BTRC)-like complex lacking CUL1, which is associated with RELA; RELA interacts directly with NFKBIA. Interacts with PRMT2. Interacts with PRKACA in platelets; this interaction is disrupted by thrombin and collagen. Interacts with HIF1AN.

Subcellular Location:
Cytoplasm. Nucleus. Note=Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export.

Post-translational modifications:
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. 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.

DISEASE:
Defects in NFKBIA are the cause of ectodermal dysplasia anhidrotic with T-cell immunodeficiency autosomal dominant (ADEDAID) [MIM:612132]. Ectodermal dysplasia defines a heterogeneous group of disorders due to abnormal development of two or more ectodermal structures. ADEDAID is an ectodermal dysplasia associated with decreased production of pro-inflammatory cytokines and certain interferons, rendering patients susceptible to infection.

Similarity:
Belongs to the NF-kappa-B inhibitor family.
Contains 5 ANK repeats.

SWISS:
P25963

Gene ID:
4792

Database links:

Entrez Gene: 4792 Human

Entrez Gene: 3635 Mouse

Entrez Gene: 25493 Rat

Omim: 112808 Human

SwissProt: P25963 Human

SwissProt: Q9Z1E3 Mouse

SwissProt: Q63746 Rat

Unigene: 81328 Human

Unigene: 170515 Mouse

Unigene: 12550 Rat

Picture

Paraformaldehyde-fixed, paraffin embedded (rat stomach); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat lymphoid); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat adrenal gland); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-IKB alpha (Tyr305) ) Polyclonal Antibody, Unconjugated (SL5515R) at 1:400 overnight at 4°C, followed by a conjugated secondary antibody (sp-0023) for 20 minutes and DAB staining.

Tissue/cell: human colon carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-NFKBIA Polyclonal Antibody, Unconjugated(SL5515R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining

Paraformaldehyde-fixed, paraffin embedded (Mouse liver); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (p-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (SL5515R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Tissue/cell:Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,SLC0005) at 37°C for 20 min; Antibody incubation with (phospho-IKB alpha (Tyr305)) polyclonal Antibody, Unconjugated (SL5515R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control: Hela.
Primary Antibody (green line): Rabbit Anti- phospho-IKB alpha (Tyr305) antibody (SL5515R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

Blank control (blue line): Hela (fixed with 70% methanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃).
Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (SL5515R),Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE,Dilution: 1μg /test.

Blank control (blue line): Jurkat (fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice).
Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (SL5515R),Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC,Dilution: 1μg /test.

Product Feedback Wall

Specific References (14) | SL5515R has been referenced in 14 publications.

[IF=2.447] Mingdong Si. et al. Gentianella acuta mitigates cardiovascular damage and inflammation in diet‑induced hypercholesterolaemic rats. Exp Ther Med. 2021 Nov;22(5):1-10 WB,IHC ; rat.

PubMed:3923527

[IF=2.185] Zhao et al. Fascin Overexpression Promotes Cholangiocarcinoma RBE Cell Proliferation, Migration, and Invasion. (2016) Technol.Cancer.Res.Trea. 15:322-33 WB ; Human.

PubMed:25882176

[IF=0] Zhang et al. Hyperbaric Oxygen and Ginkgo Biloba Extract Ameliorate Cognitive and Memory Impairment via Nuclear Factor Kappa-B Pathway in Rat Model of Alzheimer's Disease. (2015) Chin.Med.J.(Engl. 128:3088-93 WB ; Rat.

PubMed:26608991

[IF=1.69] Liu, Xinwei, et al. "Inhibition of BTK protects lungs from trauma-hemorrhagic shock-induced injury in rats." Molecular Medicine Reports 16.1 (2017): 192-200. WB ; Rat.

PubMed:28487990

[IF=4.42] Yu, Haijie, et al. "Gypenoside Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of Mitogen-Activated Protein Kinase Mediated Nuclear Factor Kappa B Pathway In Vitro and In Vivo." Frontiers in Pharmacology 7 (2016). WB ; Rat.

PubMed:27313532

[IF=2.7] Zhao, Hongyu, et al. "Betulin attenuates kidney injury in septic rats through inhibiting TLR4/NF-κB signaling pathway."Life Sciences (2016). WB ; Rat.

PubMed:26656467

[IF=1.72] Yan, Xuerui, et al. "Cyanidin-3-O-glucoside Induces Apoptosis and Inhibits Migration of Tumor Necrosis Factor-α-Treated Rat Aortic Smooth Muscle Cells." Cardiovascular Toxicology (2015): 1-9. WB ; Rat.

PubMed:2627696

[IF=1.984] Wang X et al. trans-Cinnamaldehyde Reverses Depressive-Like Behaviors in Chronic Unpredictable Mild Stress Rats by Inhibiting NF-κB/NLRP3 Inflammasome Pathway. Evid Based Complement Alternat Med. 2020 Feb 28;2020:4572185. ELISA ; rat.

PubMed:32328132

[IF=1.719] Zhou J et al. Paeonol antagonizes oncogenesis of osteosarcoma by inhibiting the function of TLR4/MAPK/NF-κB pathway. Acta Histochem. 2019 Oct 3:151455. WB ; Mouse&Human.

PubMed:31587886

[IF=1.922] Zhang et al. Apelin-13 protects against myocardial infarction-induced myocardial fibrosis. (2016) Mol.Med.Rep. 13:5262-8 IHSLCP/WB ; Rat.

PubMed:27109054

[IF=2.81] Chen et al. Naringin Alleviates Diabetic Kidney Disease through Inhibiting Oxidative Stress and Inflammatory Reaction. (2015) PLoS.On. 10:e0143868 WB ; Rat.

PubMed:26619044

[IF=2.35] Tian, Yue, et al. "Minocycline attenuates sevoflurane-induced cell injury via activation of Nrf2." International Journal of Molecular Medicine. WB ; Human.

PubMed:28260081

[IF=1.35] Gao, Wenyan, et al. "Indirubin ameliorates dextran sulfate sodium-induced ulcerative colitis in mice through the inhibition of inflammation and the induction of Foxp3-expressing regulatory T cells." Acta Histochemica (2016). WB ; Mouse.

PubMed:27396532

[IF=2.81] Li, Yuanchao, et al. "Valproate ameliorates nitroglycerin-induced migraine in trigeminal nucleus caudalis in rats through inhibition of NF-кB." The Journal of Headache and Pain 17.1 (2016): 1-9. WB ; Rat.

PubMed:27150105