Hoja de datos

Descripción biológica

Especificidad	PAX8 Antibody [J6L24] reconoce los niveles endógenos de la proteína PAX8 total.
Antecedentes	PAX8 es un factor de transcripción que pertenece a la familia de genes paired box (PAX), esencial para el desarrollo embrionario, particularmente en la glándula tiroides, el riñón, el sistema mülleriano y el timo. Estructuralmente, PAX8 contiene un dominio de unión a ADN N-terminal, que comprende una región paired box con dos subdominios (PAI y RED), un octapéptido y un homeodominio truncado, y un dominio de transactivación C-terminal. El gen PAX8, localizado en el cromosoma 2q14.1, produce seis variantes de empalme (a–f) a través de empalme alternativo, todas conservando el dominio de unión a ADN. PAX8A es la isoforma predominante en etapas embrionarias tardías y adultas. La expresión de PAX8 es específica de tejidos, encontrándose en la tiroides, el riñón, la placenta y tejidos adultos derivados del sistema mülleriano como el útero, las trompas de Falopio y el cérvix. Funcionalmente, PAX8 regula la transcripción de genes cruciales para la organogénesis, la diferenciación celular y la producción de hormonas, particularmente en la tiroides. Aunque vital durante el desarrollo, la expresión de PAX8 persiste en algunos tejidos adultos y puede adquirir funciones oncogénicas tras la transformación celular, vinculando las vías de desarrollo con la tumorigénesis.

Especificidad

PAX8 Antibody [J6L24] reconoce los niveles endógenos de la proteína PAX8 total.

Antecedentes

PAX8 es un factor de transcripción que pertenece a la familia de genes paired box (PAX), esencial para el desarrollo embrionario, particularmente en la glándula tiroides, el riñón, el sistema mülleriano y el timo. Estructuralmente, PAX8 contiene un dominio de unión a ADN N-terminal, que comprende una región paired box con dos subdominios (PAI y RED), un octapéptido y un homeodominio truncado, y un dominio de transactivación C-terminal. El gen PAX8, localizado en el cromosoma 2q14.1, produce seis variantes de empalme (a–f) a través de empalme alternativo, todas conservando el dominio de unión a ADN. PAX8A es la isoforma predominante en etapas embrionarias tardías y adultas. La expresión de PAX8 es específica de tejidos, encontrándose en la tiroides, el riñón, la placenta y tejidos adultos derivados del sistema mülleriano como el útero, las trompas de Falopio y el cérvix. Funcionalmente, PAX8 regula la transcripción de genes cruciales para la organogénesis, la diferenciación celular y la producción de hormonas, particularmente en la tiroides. Aunque vital durante el desarrollo, la expresión de PAX8 persiste en algunos tejidos adultos y puede adquirir funciones oncogénicas tras la transformación celular, vinculando las vías de desarrollo con la tumorigénesis.

Información de uso

Aplicación

WB, IP, IHC, FCM

Dilución

WB	IP	IHC	FCM
1:50-1:500	1:100	1:50 - 1:100	1:100

Reactividad

Human, Mouse, Rat

Fuente

Mouse Monoclonal Antibody

MW

48 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/Nuclear 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/Nuclear 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/Nuclear 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 1:50), 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 1389. 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 60s is recommended)

Métodos experimentales

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear 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/Nuclear 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/Nuclear 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 1:50), 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

1389. 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 60s is recommended)

Referencias

https://pubmed.ncbi.nlm.nih.gov/35806410/

Datos de aplicación

IHC

Validado por Selleck

Immunohistochemical analysis of formalin fixed paraffin embedded mouse kidney tissue with F2154 at 1:20 dilution.

WB

Validado por Selleck

Lane 1: Hela