Datasheet

Biological Description

Specificity	ITCH Rabbit mAb recognizes endogenous levels of total ITCH protein.
Background	ITCH is a HECT domain-containing E3 ubiquitin ligase initially identified in genetic studies of the mouse agouti locus, where mutations result in distinct coat color changes. One particular agouti mutation, known as non-agouti-lethal 18H, is notable for causing immunological defects not seen in other agouti mutant mice. This 18H mutation, which leads to a darker coat color, is caused by a radiation-induced chromosomal inversion that deletes 18 and 20 base pairs from the proximal and distal inversion breakpoints, respectively, disrupting the expression of both the agouti and Itch genes. The Itch gene encodes a protein of 854 amino acids with a molecular weight of approximately 113 kDa. ITCH functions as a monomeric E3 ubiquitin ligase belonging to the HECT-type family, characterized by a modular structure that includes an N-terminal Ca2+-dependent phospholipid-binding C2 domain, multiple WW domains involved in protein-protein interactions, and a C-terminal HECT domain. The HECT domain interacts with a specific E2 ubiquitin-conjugating enzyme and contains a highly conserved cysteine residue that forms a thioester bond with ubiquitin before transferring ubiquitin to target proteins. ITCH contains four WW domains and a distinctive proline-rich region (PRR) between residues 195 and 246, which plays a crucial role in its regulatory functions. ITCH-mediated ubiquitination extends beyond immune signaling pathways, targeting key regulators in the Hedgehog, Wnt/β-catenin, Hippo, and Notch signaling pathways, highlighting its broad impact on cellular signaling.

Specificity

ITCH Rabbit mAb recognizes endogenous levels of total ITCH protein.

Background

ITCH is a HECT domain-containing E3 ubiquitin ligase initially identified in genetic studies of the mouse agouti locus, where mutations result in distinct coat color changes. One particular agouti mutation, known as non-agouti-lethal 18H, is notable for causing immunological defects not seen in other agouti mutant mice. This 18H mutation, which leads to a darker coat color, is caused by a radiation-induced chromosomal inversion that deletes 18 and 20 base pairs from the proximal and distal inversion breakpoints, respectively, disrupting the expression of both the agouti and Itch genes. The Itch gene encodes a protein of 854 amino acids with a molecular weight of approximately 113 kDa. ITCH functions as a monomeric E3 ubiquitin ligase belonging to the HECT-type family, characterized by a modular structure that includes an N-terminal Ca2+-dependent phospholipid-binding C2 domain, multiple WW domains involved in protein-protein interactions, and a C-terminal HECT domain. The HECT domain interacts with a specific E2 ubiquitin-conjugating enzyme and contains a highly conserved cysteine residue that forms a thioester bond with ubiquitin before transferring ubiquitin to target proteins. ITCH contains four WW domains and a distinctive proline-rich region (PRR) between residues 195 and 246, which plays a crucial role in its regulatory functions. ITCH-mediated ubiquitination extends beyond immune signaling pathways, targeting key regulators in the Hedgehog, Wnt/β-catenin, Hippo, and Notch signaling pathways, highlighting its broad impact on cellular signaling.

Usage Information

Application

WB, IP

Dilution

WB	IP
1:1000	1:200

Reactivity

Human, Mouse, Rat

Source

Rabbit

MW

105 kDa

Storage Buffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

Storage
(from the date of receipt)

–20°C (avoid freeze-thaw cycles), 2 years

Exprimental Methods
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/SDS/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/SDS/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/SDS/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 816. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Exprimental Methods

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/SDS/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/SDS/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/SDS/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

816. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

References

[1] Melino G, et al. Cell Death Differ. 2008 Jul;15(7):1103-12.

Application Data

WB

Validated by Selleck

Lane 1: Ramos
Lane 2: Raji
Lane 3: NIH/3T3