Fiche technique

Description biologique

Spécificité	ITCH Antibody [K4F16] reconnaît les niveaux endogènes de la protéine ITCH totale.
Contexte	ITCH est une ubiquitine ligase E3 contenant un domaine HECT, initialement identifiée dans des études génétiques du locus agouti de la souris, où des mutations entraînent des changements distincts de couleur de pelage. Une mutation agouti particulière, connue sous le nom de non-agouti-léthal 18H, est notable pour causer des défauts immunologiques non observés chez d'autres souris mutantes agouti. Cette mutation 18H, qui entraîne une couleur de pelage plus foncée, est causée par une inversion chromosomique induite par rayonnement qui supprime respectivement 18 et 20 paires de bases des points de rupture d'inversion proximaux et distaux, perturbant l'expression des gènes agouti et Itch. Le gène Itch code une protéine de 854 acides aminés avec un poids moléculaire d'environ 113 kDa. ITCH fonctionne comme une ubiquitine ligase E3 monomérique appartenant à la famille de type HECT, caractérisée par une structure modulaire qui comprend un domaine C2 de liaison aux phospholipides dépendant du Ca2+ N-terminal, de multiples domaines WW impliqués dans les interactions protéine-protéine, et un domaine HECT C-terminal. Le domaine HECT interagit avec une enzyme de conjugaison d'ubiquitine E2 spécifique et contient un résidu cystéine hautement conservé qui forme une liaison thioester avec l'ubiquitine avant de transférer l'ubiquitine aux protéines cibles. ITCH contient quatre domaines WW et une région riche en proline (PRR) distinctive entre les résidus 195 et 246, qui joue un rôle crucial dans ses fonctions régulatrices. L'ubiquitination médiatisée par ITCH s'étend au-delà des voies de signalisation immunitaires, ciblant les régulateurs clés des voies de signalisation Hedgehog, Wnt/β-caténine, Hippo et Notch, soulignant son impact large sur la signalisation cellulaire.

Spécificité

ITCH Antibody [K4F16] reconnaît les niveaux endogènes de la protéine ITCH totale.

Contexte

ITCH est une ubiquitine ligase E3 contenant un domaine HECT, initialement identifiée dans des études génétiques du locus agouti de la souris, où des mutations entraînent des changements distincts de couleur de pelage. Une mutation agouti particulière, connue sous le nom de non-agouti-léthal 18H, est notable pour causer des défauts immunologiques non observés chez d'autres souris mutantes agouti. Cette mutation 18H, qui entraîne une couleur de pelage plus foncée, est causée par une inversion chromosomique induite par rayonnement qui supprime respectivement 18 et 20 paires de bases des points de rupture d'inversion proximaux et distaux, perturbant l'expression des gènes agouti et Itch. Le gène Itch code une protéine de 854 acides aminés avec un poids moléculaire d'environ 113 kDa. ITCH fonctionne comme une ubiquitine ligase E3 monomérique appartenant à la famille de type HECT, caractérisée par une structure modulaire qui comprend un domaine C2 de liaison aux phospholipides dépendant du Ca2+ N-terminal, de multiples domaines WW impliqués dans les interactions protéine-protéine, et un domaine HECT C-terminal. Le domaine HECT interagit avec une enzyme de conjugaison d'ubiquitine E2 spécifique et contient un résidu cystéine hautement conservé qui forme une liaison thioester avec l'ubiquitine avant de transférer l'ubiquitine aux protéines cibles. ITCH contient quatre domaines WW et une région riche en proline (PRR) distinctive entre les résidus 195 et 246, qui joue un rôle crucial dans ses fonctions régulatrices. L'ubiquitination médiatisée par ITCH s'étend au-delà des voies de signalisation immunitaires, ciblant les régulateurs clés des voies de signalisation Hedgehog, Wnt/β-caténine, Hippo et Notch, soulignant son impact large sur la signalisation cellulaire.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:200

Réactivité

Human, Mouse, Rat

Source

Rabbit Monoclonal Antibody

MW

105 kDa

Tampon de stockage

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

Stockage
(À partir de la date de réception)

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

Méthodes expérimentales
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.

Méthodes expérimentales

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.

Références

https://pubmed.ncbi.nlm.nih.gov/18552861/

Données dapplication

WB

Validé par Selleck

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