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Descripción biológica

Especificidad	GCLC Antibody [C5F2] reconoce los niveles endógenos de proteína GCLC total.
Antecedentes	La subunidad catalítica de la glutamato-cisteína ligasa (GCLC) es una enzima clave implicada en la biosíntesis de Glutathione (GSH), un antioxidante esencial que ayuda a proteger las células del daño oxidativo y mantiene el equilibrio redox celular. GCLC forma parte de un complejo enzimático heterodimérico, GCL, que consta de GCLC y la subunidad modificadora GCLM. La enzima tiene un sitio activo altamente conservado responsable de catalizar el primer paso y limitante de la velocidad en la síntesis de GSH, que implica la ligación de glutamato y cisteína para formar γ-glutamilcisteína. Su función principal es catalizar la formación de γ-glutamilcisteína, un precursor de GSH. Este proceso es vital para la defensa celular contra el estrés oxidativo, la desintoxicación y el mantenimiento de la homeostasis redox intracelular. GCLC, en combinación con GCLM, regula la tasa de producción de GSH, lo que la convierte en una enzima crucial para mantener la función celular adecuada. La actividad de GCLC está estrechamente regulada por su interacción con GCLM. GCLM mejora la eficiencia catalítica de GCLC, y la actividad de la enzima puede modularse en respuesta al estrés oxidativo y a los cambios en los estados metabólicos. Además, la expresión de GCLC se regula al alza durante el estrés oxidativo para satisfacer la mayor demanda de GSH. También está implicada en la desintoxicación, la modulación de la respuesta inmunitaria y la protección contra las especies reactivas de oxígeno (ROS). La desregulación de la actividad de GCLC y la producción de GSH se asocia con enfermedades neurodegenerativas como la enfermedad de Parkinson, donde el estrés oxidativo es una característica prominente.

Especificidad

GCLC Antibody [C5F2] reconoce los niveles endógenos de proteína GCLC total.

Antecedentes

La subunidad catalítica de la glutamato-cisteína ligasa (GCLC) es una enzima clave implicada en la biosíntesis de Glutathione (GSH), un antioxidante esencial que ayuda a proteger las células del daño oxidativo y mantiene el equilibrio redox celular. GCLC forma parte de un complejo enzimático heterodimérico, GCL, que consta de GCLC y la subunidad modificadora GCLM. La enzima tiene un sitio activo altamente conservado responsable de catalizar el primer paso y limitante de la velocidad en la síntesis de GSH, que implica la ligación de glutamato y cisteína para formar γ-glutamilcisteína. Su función principal es catalizar la formación de γ-glutamilcisteína, un precursor de GSH. Este proceso es vital para la defensa celular contra el estrés oxidativo, la desintoxicación y el mantenimiento de la homeostasis redox intracelular. GCLC, en combinación con GCLM, regula la tasa de producción de GSH, lo que la convierte en una enzima crucial para mantener la función celular adecuada. La actividad de GCLC está estrechamente regulada por su interacción con GCLM. GCLM mejora la eficiencia catalítica de GCLC, y la actividad de la enzima puede modularse en respuesta al estrés oxidativo y a los cambios en los estados metabólicos. Además, la expresión de GCLC se regula al alza durante el estrés oxidativo para satisfacer la mayor demanda de GSH. También está implicada en la desintoxicación, la modulación de la respuesta inmunitaria y la protección contra las especies reactivas de oxígeno (ROS). La desregulación de la actividad de GCLC y la producción de GSH se asocia con enfermedades neurodegenerativas como la enfermedad de Parkinson, donde el estrés oxidativo es una característica prominente.

Información de uso

Aplicación

WB, IP

Dilución

WB	IP
1:20000	1:50

Reactividad

Human, Mouse, Rat

Fuente

Rabbit Monoclonal Antibody

MW

73 kDa

Tampón de almacenamiento

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

Almacenamiento
(Desde la fecha de recepción)

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

Métodos experimentales
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/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/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/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: 10%. 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 1:20000), 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 1300. 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. (Exposure time of at least 90s is recommended)

Métodos experimentales

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/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/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/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: 10%. 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 1:20000), 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

1300. 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. (Exposure time of at least 90s is recommended)

Referencias

https://pubmed.ncbi.nlm.nih.gov/16081425/

Datos de aplicación

WB

Validado por Selleck

Lane 1: Jurkat
Lane 2: C6
Lane 3: NIH/3T3