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Descripción biológica

Especificidad	Na+/H+ Exchanger-1 Antibody [K9D3] detecta los niveles endógenos de la proteína total Na+/H+ Exchanger-1.
Antecedentes	Na+/H+ Exchanger-1 (NHE1, SLC9A1) es un antiportador de membrana integral expresado ubicuamente que desempeña un papel central en la regulación de la homeostasis del pH intracelular (pHi). NHE1 está compuesto por 12 hélices transmembrana (TMHs) con ambos N- y C-terminales orientados hacia el citosol. Los segmentos de transporte clave incluyen TM IV, VII y IX, junto con los bucles reentrantes IL2 e IL4, y los sitios de glicosilación en el bucle extracelular 5 (EL5) que confieren estabilidad estructural. El dominio citoplasmático C-terminal extendido de ~315 residuos contiene numerosos sitios de fosforilación de serina/treonina y motivos de unión a ezrina (residuos 553–564), anclando NHE1 al citoesqueleto de actina. NHE1 se activa alostéricamente por la acidificación intracelular, lo que promueve la unión de protones a un sitio modificador no transportador (coeficiente de Hill ~3), desencadenando cambios conformacionales de estados orientados hacia adentro a estados orientados hacia afuera a través de la inclinación de la hélice y vías de acceso llenas de agua, similar al transportador bacteriano NhaA. Media el intercambio electrogénico de Na+ extracelular (Km 5–50 mM) por H+ intracelular en una estequiometría 1:1, utilizando el gradiente transmembrana de Na+ como su fuerza impulsora sin aporte directo de energía. Esto permite una rápida recuperación del pHi después de la acidosis, la regulación del volumen celular mediante la entrada de Na+ y el andamiaje de la protrusión de lamelipodios a través de interacciones ERM-actina. La señalización hormonal y de factores de crecimiento puede fosforilar serinas C-terminales (p. ej., S703, Thr653) a través de las vías NHERF1, ERK y PKA, desplazando el punto de ajuste del pHi hacia la alcalinidad. Patológicamente, en la lesión por isquemia-reperfusión, el NHE1 activado por ácido promueve una sobrecarga citotóxica de Na+/Ca2+ a través de la actividad de NCX en modo inverso, exacerbando el infarto de miocardio. La regulación al alza crónica de NHE1 también está implicada en la invasión tumoral a través de la alcalinización pericelular y la remodelación de la matriz, así como en la hipertrofia cardíaca.

Especificidad

Na+/H+ Exchanger-1 Antibody [K9D3] detecta los niveles endógenos de la proteína total Na+/H+ Exchanger-1.

Antecedentes

Na+/H+ Exchanger-1 (NHE1, SLC9A1) es un antiportador de membrana integral expresado ubicuamente que desempeña un papel central en la regulación de la homeostasis del pH intracelular (pHi). NHE1 está compuesto por 12 hélices transmembrana (TMHs) con ambos N- y C-terminales orientados hacia el citosol. Los segmentos de transporte clave incluyen TM IV, VII y IX, junto con los bucles reentrantes IL2 e IL4, y los sitios de glicosilación en el bucle extracelular 5 (EL5) que confieren estabilidad estructural. El dominio citoplasmático C-terminal extendido de ~315 residuos contiene numerosos sitios de fosforilación de serina/treonina y motivos de unión a ezrina (residuos 553–564), anclando NHE1 al citoesqueleto de actina. NHE1 se activa alostéricamente por la acidificación intracelular, lo que promueve la unión de protones a un sitio modificador no transportador (coeficiente de Hill ~3), desencadenando cambios conformacionales de estados orientados hacia adentro a estados orientados hacia afuera a través de la inclinación de la hélice y vías de acceso llenas de agua, similar al transportador bacteriano NhaA. Media el intercambio electrogénico de Na+ extracelular (Km 5–50 mM) por H+ intracelular en una estequiometría 1:1, utilizando el gradiente transmembrana de Na+ como su fuerza impulsora sin aporte directo de energía. Esto permite una rápida recuperación del pHi después de la acidosis, la regulación del volumen celular mediante la entrada de Na+ y el andamiaje de la protrusión de lamelipodios a través de interacciones ERM-actina. La señalización hormonal y de factores de crecimiento puede fosforilar serinas C-terminales (p. ej., S703, Thr653) a través de las vías NHERF1, ERK y PKA, desplazando el punto de ajuste del pHi hacia la alcalinidad. Patológicamente, en la lesión por isquemia-reperfusión, el NHE1 activado por ácido promueve una sobrecarga citotóxica de Na+/Ca2+ a través de la actividad de NCX en modo inverso, exacerbando el infarto de miocardio. La regulación al alza crónica de NHE1 también está implicada en la invasión tumoral a través de la alcalinización pericelular y la remodelación de la matriz, así como en la hipertrofia cardíaca.

Información de uso

Aplicación

WB

Dilución

WB

1:500

Reactividad

Mouse, Human, Amphibian, Fish, Avian, Vertebrates

Fuente

Mouse Monoclonal Antibody

MW

~100-110 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. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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 1:500), 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 1. 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.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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 1:500), 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

1. 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/34108458/
https://pubmed.ncbi.nlm.nih.gov/33273619/

Datos de aplicación

WB

Validado por Selleck

Lane 1: U87MG, Lane 2: THP-1, Lane 3: 22RV1, Lane 4: Mouse brain