PRODUCT CODE: ER1901-94

ITCH Rabbit Polyclonal Antibody (ER1901-94)

Applications

  • WB

  • IHC-P

  • ICC

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

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Western blot analysis of ITCH on Siha cell  lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-94, 1/2000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of ITCH on Siha cell  lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-94, 1/2000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of ITCH on Mouse pancreatic tissue lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-94, 1/1000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • ICC staining of A431 in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ER1901-94, 1/200) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining of ITCH in LOVO cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ER1901-94, 1/200) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining of ITCH in Siha cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ER1901-94, 1/200) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
  • Immunohistochemical analysis of paraffin-embedded Human liver cancer tissue using anti-ITCH antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-94, 1/50) for 30 minutes 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.
  • Immunohistochemical analysis of paraffin-embedded mouse pancreas tissue using anti-ITCH antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-94, 1/50) for 30 minutes 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.
  • Flow cytometric analysis of ITCH was done on LOVO cells. The cells were fixed, permeabilized and stained with the primary antibody (ER1901-94, 1/100) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated goat anti-rabbit IgG Secondary antibody at 1/500 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; green).
Western blot analysis of ITCH on Siha cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-94, 1/2000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.

Applications

  • WB

  • IHC-P

  • ICC

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Rabbit polyclonal primary

Product Name

ITCH Rabbit Polyclonal Antibody (ER1901-94)

Immunogen

Recombinant protein within human itch aa 300-500.

Host

Rabbit

Positive Control

Siha cell lysates, Mouse pancreatic tissue lysates, Hela, LOVO, Siha, Human liver cancer tissue, rat brain tissue, mouse pancreas tissue.

Conjugation

Unconjugated

Clonality

Polyclonal

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

1 ug/ul

PURIFICATION

Protein A purified.

MOLECULAR WEIGHT

Predicted band size 102/98/86 kDa.

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:1000-1:5000

  • IHC-P

  • 1:50-1:200

  • ICC

  • 1:100-1:500

  • FC

  • 1:50-1:100

TARGET

UNIPROT #

PROTEIN NAME

ITCH

SYNONYMS

ADMFD antibody; AIF4 antibody; AIP4 antibody; Atrophin 1 interacting protein 4 antibody; Atrophin-1-interacting protein 4 antibody; dJ468O1.1 antibody; dJ468O1.1 (atrophin 1 interacting protein 4 (AIP4)) antibody; dJ468O1.1 atrophin 1 interacting protein 4 AIP4 antibody; E3 ubiquitin protein ligase Itchy homolog antibody; E3 ubiquitin-protein ligase Itchy homolog antibody; EC 6.3.2 antibody; Itch antibody; ITCH_HUMAN antibody; Itchy E3 ubiquitin protein ligase antibody; Itchy E3 ubiquitin protein ligase homolog antibody; Itchy E3 ubiquitin protein ligase homolog mouse antibody; Itchy E3 ubiquitin protein ligase, mouse, homolog of antibody; Itchy homolog E3 ubiquitin protein ligase antibody; Itchy mouse homolog E3 ubiquitin protein ligase antibody; NAPP1 antibody; NFE2 associated polypeptide 1 antibody; NFE2-associated polypeptide 1 antibody; Ubiquitin protein ligase ITCH antibody

TISSUE SPECIFICITY

Widely expressed.

POST-TRANSLATIONAL MODIFICATION

On T-cell activation, phosphorylation by the JNK cascade on serine and threonine residues surrounding the PRR domain accelerates the ubiquitination and degradation of JUN and JUNB. The increased ITCH catalytic activity due to phosphorylation by JNK1 may occur due to a conformational change disrupting the interaction between the PRR/WW motifs domain and the HECT domain and, thus exposing the HECT domain (By similarity). Phosphorylation by FYN reduces interaction with JUNB and negatively controls JUN ubiquitination and degradation.; Monoubiquitinated. Autopolyubiquitinated with 'Lys-63' linkages which does not lead to protein degradation.

SUBCELLULAR LOCATION

Cell membrane, Cytoplasm, Endosome, Membrane, Nucleus.

FUNCTION

Acts as an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates . Involved in the control of inflammatory signaling pathways . Essential component of a ubiquitin-editing protein complex, comprising also TNFAIP3, TAX1BP1 and RNF11, that ensures the transient nature of inflammatory signaling pathways . Promotes the association of the complex after TNF stimulation . Once the complex is formed, TNFAIP3 deubiquitinates 'Lys-63' polyubiquitin chains on RIPK1 and catalyzes the formation of 'Lys-48'-polyubiquitin chains . This leads to RIPK1 proteasomal degradation and consequently termination of the TNF- or LPS-mediated activation of NFKB1. Ubiquitinates RIPK2 by 'Lys-63'-linked conjugation and influences NOD2-dependent signal transduction pathways. Regulates the transcriptional activity of several transcription factors, and probably plays an important role in the regulation of immune response . Ubiquitinates NFE2 by 'Lys-63' linkages and is implicated in the control of the development of hematopoietic lineages .