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Descripción biológica

Especificidad	Pannexin-1 Antibody [C19F23] reconoce los niveles endógenos de la proteína pannexin-1 total. Este anticuerpo detecta un fragmento de pannexin-1 amino-terminal producido por el clivaje de caspasa.
Antecedentes	Las proteínas de la familia Pannexin forman grandes canales transmembrana porosos, permitiendo la liberación de ATP al medio extracelular y están involucradas en varios procesos biológicos, incluyendo inflamación, señalización de ATP, plasticidad sináptica y neurotoxicidad. La familia incluye tres miembros: Panx1 (426 aminoácidos, 47.6 kDa), Panx2 (664 aminoácidos, 73.3 kDa) y Panx3 (392 aminoácidos, 44.7 kDa), pertenecientes a la superfamilia de uniones no-gap en vertebrados. Panx1 forma un canal selectivo de aniones que permite el paso de iones y moléculas de hasta 1 kDa, incluyendo ATP, Ca++ intracelular y glucosa. La liberación de ATP mediada por Panx1 es crucial para condiciones fisiológicas y fisiopatológicas y la señalización purinérgica. Panx1, activado por la despolarización de la membrana y el estiramiento mecánico, interactúa con receptores purinérgicos como P2X7 y regula las funciones inmunes liberando ATP y UTP como señales de "búsqueme" para eliminar las células apoptóticas. Panx1 se expresa principalmente en la membrana plasmática y está implicado en varias patologías, actuando potencialmente como un supresor tumoral o influyendo en la muerte celular isquémica, la aterosclerosis, la apoptosis, el VIH y las convulsiones.

Especificidad

Pannexin-1 Antibody [C19F23] reconoce los niveles endógenos de la proteína pannexin-1 total. Este anticuerpo detecta un fragmento de pannexin-1 amino-terminal producido por el clivaje de caspasa.

Antecedentes

Las proteínas de la familia Pannexin forman grandes canales transmembrana porosos, permitiendo la liberación de ATP al medio extracelular y están involucradas en varios procesos biológicos, incluyendo inflamación, señalización de ATP, plasticidad sináptica y neurotoxicidad. La familia incluye tres miembros: Panx1 (426 aminoácidos, 47.6 kDa), Panx2 (664 aminoácidos, 73.3 kDa) y Panx3 (392 aminoácidos, 44.7 kDa), pertenecientes a la superfamilia de uniones no-gap en vertebrados. Panx1 forma un canal selectivo de aniones que permite el paso de iones y moléculas de hasta 1 kDa, incluyendo ATP, Ca++ intracelular y glucosa. La liberación de ATP mediada por Panx1 es crucial para condiciones fisiológicas y fisiopatológicas y la señalización purinérgica. Panx1, activado por la despolarización de la membrana y el estiramiento mecánico, interactúa con receptores purinérgicos como P2X7 y regula las funciones inmunes liberando ATP y UTP como señales de "búsqueme" para eliminar las células apoptóticas. Panx1 se expresa principalmente en la membrana plasmática y está implicado en varias patologías, actuando potencialmente como un supresor tumoral o influyendo en la muerte celular isquémica, la aterosclerosis, la apoptosis, el VIH y las convulsiones.

Información de uso

Aplicación

WB, IP

Dilución

WB	IP
1:1000	1:100

Reactividad

Human, Mouse, Rat

Fuente

Rabbit Monoclonal Antibody

MW

45-55, 19 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 1:1000), 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 646. 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.

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 1:1000), 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

646. 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.

Referencias

https://pubmed.ncbi.nlm.nih.gov/33394272/

Datos de aplicación

WB

Validado por Selleck

Lane 1: PANC-1
Lane 2: K-562
Lane 3: U87
Lane 4: 786-0
Lane 5: C2C12
Lane 6: A20
Lane 7: L-929
Lane 8: RBL-2H3