Scheda tecnica

Descrizione biologica

Specificità	ITCH Antibody [K4F16] riconosce i livelli endogeni della proteina ITCH totale.
Contesto	ITCH è una E3 ubiquitina ligasi contenente un dominio HECT, inizialmente identificata in studi genetici del locus agouti del topo, dove le mutazioni portano a distinti cambiamenti nel colore del mantello. Una particolare mutazione agouti, nota come non-agouti-letale 18H, è notevole per aver causato difetti immunologici non osservati in altri topi mutanti agouti. Questa mutazione 18H, che porta a un colore del mantello più scuro, è causata da un'inversione cromosomica indotta da radiazioni che delega 18 e 20 coppie di basi dai punti di rottura di inversione prossimale e distale, rispettivamente, interrompendo l'espressione di entrambi i geni agouti e Itch. Il gene Itch codifica una proteina di 854 amminoacidi con un peso molecolare di circa 113 kDa. ITCH funziona come una E3 ubiquitina ligasi monomerica appartenente alla famiglia di tipo HECT, caratterizzata da una struttura modulare che include un dominio C2 N-terminale di legame ai fosfolipidi dipendente dal Ca2+, domini WW multipli coinvolti nelle interazioni proteina-proteina e un dominio HECT C-terminale. Il dominio HECT interagisce con un enzima di coniugazione ubiquitina E2 specifico e contiene un residuo di cisteina altamente conservato che forma un legame tioestere con l'ubiquitina prima di trasferire l'ubiquitina alle proteine bersaglio. ITCH contiene quattro domini WW e una distintiva regione ricca di prolina (PRR) tra i residui 195 e 246, che svolge un ruolo cruciale nelle sue funzioni regolatorie. L'ubiquitinazione mediata da ITCH si estende oltre le vie di segnalazione immunitaria, prendendo di mira regolatori chiave nelle vie di segnalazione Hedgehog, Wnt/β-catenina, Hippo e Notch, evidenziando il suo ampio impatto sulla segnalazione cellulare.

Specificità

ITCH Antibody [K4F16] riconosce i livelli endogeni della proteina ITCH totale.

Contesto

ITCH è una E3 ubiquitina ligasi contenente un dominio HECT, inizialmente identificata in studi genetici del locus agouti del topo, dove le mutazioni portano a distinti cambiamenti nel colore del mantello. Una particolare mutazione agouti, nota come non-agouti-letale 18H, è notevole per aver causato difetti immunologici non osservati in altri topi mutanti agouti. Questa mutazione 18H, che porta a un colore del mantello più scuro, è causata da un'inversione cromosomica indotta da radiazioni che delega 18 e 20 coppie di basi dai punti di rottura di inversione prossimale e distale, rispettivamente, interrompendo l'espressione di entrambi i geni agouti e Itch. Il gene Itch codifica una proteina di 854 amminoacidi con un peso molecolare di circa 113 kDa. ITCH funziona come una E3 ubiquitina ligasi monomerica appartenente alla famiglia di tipo HECT, caratterizzata da una struttura modulare che include un dominio C2 N-terminale di legame ai fosfolipidi dipendente dal Ca2+, domini WW multipli coinvolti nelle interazioni proteina-proteina e un dominio HECT C-terminale. Il dominio HECT interagisce con un enzima di coniugazione ubiquitina E2 specifico e contiene un residuo di cisteina altamente conservato che forma un legame tioestere con l'ubiquitina prima di trasferire l'ubiquitina alle proteine bersaglio. ITCH contiene quattro domini WW e una distintiva regione ricca di prolina (PRR) tra i residui 195 e 246, che svolge un ruolo cruciale nelle sue funzioni regolatorie. L'ubiquitinazione mediata da ITCH si estende oltre le vie di segnalazione immunitaria, prendendo di mira regolatori chiave nelle vie di segnalazione Hedgehog, Wnt/β-catenina, Hippo e Notch, evidenziando il suo ampio impatto sulla segnalazione cellulare.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:1000	1:200

Reattività

Human, Mouse, Rat

Fonte

Rabbit Monoclonal Antibody

MW

105 kDa

Tampone di conservazione

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

Conservazione
(Dalla data di ricevimento)

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

Metodi sperimentali
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.

Metodi sperimentali

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.

Riferimenti

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

Dati di applicazione

WB

Validato da Selleck

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