ER1901-94

ITCH Rabbit Polyclonal Antibody

cat.: ER1901-94

Product Type:	Rabbit polyclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IHC-P, IF-Cell, FC
Clonality:	Polyclonal

Form:	Liquid
Storage condition:	Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer:	1*PBS (pH7.4), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size 102/98/86 kDa.
Isotype:	IgG
Immunogen:	Recombinant protein within Human ITCH aa 302-481 / 903.
Positive control:	Mouse brain tissue lysate, Hela, LOVO, Siha, Human liver cancer tissue, rat brain tissue, mouse pancreas tissue.
Subcellular location:	Cell membrane, Cytoplasm, Endosome, Membrane, Nucleus.
Recommended Dilutions: WB IHC-P IF-Cell FC	1:1000-1:5000 1:50-1:200 1:100-1:500 1:50-1:100
Uniprot #:	SwissProt: Q96J02 Human \| Q8C863 Mouse \| A0A8I5ZZU1 Rat
Alternative names:	ADMFD AIF4 AIP4 Atrophin 1 interacting protein 4 Atrophin-1-interacting protein 4 dJ468O1.1 dJ468O1.1 (atrophin 1 interacting protein 4 (AIP4)) dJ468O1.1 atrophin 1 interacting protein 4 AIP4 E3 ubiquitin protein ligase Itchy homolog E3 ubiquitin-protein ligase Itchy homolog EC 6.3.2 Itch ITCH_HUMAN Itchy E3 ubiquitin protein ligase Itchy E3 ubiquitin protein ligase homolog Itchy E3 ubiquitin protein ligase homolog mouse Itchy E3 ubiquitin protein ligase, mouse, homolog of Itchy homolog E3 ubiquitin protein ligase Itchy mouse homolog E3 ubiquitin protein ligase NAPP1 NFE2 associated polypeptide 1 NFE2-associated polypeptide 1 Ubiquitin protein ligase ITCH

Images

Fig1: Western blot analysis of ITCH on different lysates with Rabbit anti-ITCH antibody (ER1901-94) at 1/5,000 dilution.

Lane 1: Mouse brain tissue lysate

Lysates/proteins at 15 µg/Lane.
Exposure time: 1 minutes; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: ER1901-94, 1/5,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 102 kDa
Observed band size: 102 kDa

Fig2: Western blot analysis of ITCH on different lysates with Rabbit anti-ITCH antibody (ER1901-94) at 1/2,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-ITCH KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 102 kDa
Observed band size: 102 kDa

Exposure time: 10 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 (ER1901-94) at 1/2,000 dilution was used in 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.

	Fig3: ICC staining of ITCH in A431 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).
	Fig4: 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).
	Fig5: 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).
	Fig6: 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.

Fig7: 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.

Fig8: 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).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.