Datasheet

Biologische Beschrijving

Specificiteit	Na+/H+ Exchanger-1 Antibody [K9D3] detecteert endogene niveaus van totaal Na+/H+ Exchanger-1 eiwit.
Achtergrond	Na+/H+ Exchanger-1 (NHE1, SLC9A1) is een ubiquitair tot expressie gebracht integraal membraan-antiporter die een centrale rol speelt bij het reguleren van de intracellulaire pH (pHi) homeostase. NHE1 is samengesteld uit 12 transmembraanhelices (TMHs) met zowel de N- als de C-termini naar het cytosol gericht. Belangrijke transportsegmenten zijn TM IV, VII en IX, samen met re-entrante lussen IL2 en IL4, en glycosyleringsplaatsen op extracellulaire lus 5 (EL5) die structurele stabiliteit verlenen. Het verlengde, ~315 residuen tellende C-terminale cytoplasmatische domein bevat talrijke serine/threonine fosforyleringsplaatsen en ezrine-bindende motieven (residuen 553–564), die NHE1 verankeren aan het actine-cytoskelet. NHE1 wordt allosterisch geactiveerd door intracellulaire verzuring, wat protonbinding aan een niet-transport modificatiesite (Hill-coëfficiënt ~3) bevordert, en conformationele veranderingen van naar binnen- naar naar buiten-gerichte toestanden teweegbrengt via helixkanteling en met water gevulde toegangspaden, vergelijkbaar met de bacteriële NhaA-transporter. Het medieert elektrogene uitwisseling van extracellulair Na+ (Km 5–50 mM) voor intracellulair H+ in een 1:1 stoichiometrie, waarbij de transmembraan Na+-gradiënt als drijvende kracht wordt gebruikt zonder directe energie-input. Dit maakt een snel herstel van pHi na acidose, regulatie van celvolume via Na+-influx, en steigerbouw van lamellipodia-uitstulpingen via ERM-actine-interacties mogelijk. Hormonale en groeifactor-signalering kan C-terminale serines (bijv. S703, Thr653) fosforyleren via NHERF1-, ERK- en PKA-routes, waardoor het pHi-setpoint verschuift naar alkaliteit. Pathologisch, bij ischemie-reperfusieletsel, bevordert zuur-geactiveerd NHE1 cytotoxische Na+/Ca2+-overbelasting via reverse-mode NCX-activiteit, wat myocardinfarct verergert. Chronische NHE1-upregulatie is ook betrokken bij tumorinvasie via pericellulaire alkalisering en matrixremodellering, evenals bij harthypertrofie.

Specificiteit

Na+/H+ Exchanger-1 Antibody [K9D3] detecteert endogene niveaus van totaal Na+/H+ Exchanger-1 eiwit.

Achtergrond

Na+/H+ Exchanger-1 (NHE1, SLC9A1) is een ubiquitair tot expressie gebracht integraal membraan-antiporter die een centrale rol speelt bij het reguleren van de intracellulaire pH (pHi) homeostase. NHE1 is samengesteld uit 12 transmembraanhelices (TMHs) met zowel de N- als de C-termini naar het cytosol gericht. Belangrijke transportsegmenten zijn TM IV, VII en IX, samen met re-entrante lussen IL2 en IL4, en glycosyleringsplaatsen op extracellulaire lus 5 (EL5) die structurele stabiliteit verlenen. Het verlengde, ~315 residuen tellende C-terminale cytoplasmatische domein bevat talrijke serine/threonine fosforyleringsplaatsen en ezrine-bindende motieven (residuen 553–564), die NHE1 verankeren aan het actine-cytoskelet. NHE1 wordt allosterisch geactiveerd door intracellulaire verzuring, wat protonbinding aan een niet-transport modificatiesite (Hill-coëfficiënt ~3) bevordert, en conformationele veranderingen van naar binnen- naar naar buiten-gerichte toestanden teweegbrengt via helixkanteling en met water gevulde toegangspaden, vergelijkbaar met de bacteriële NhaA-transporter. Het medieert elektrogene uitwisseling van extracellulair Na+ (Km 5–50 mM) voor intracellulair H+ in een 1:1 stoichiometrie, waarbij de transmembraan Na+-gradiënt als drijvende kracht wordt gebruikt zonder directe energie-input. Dit maakt een snel herstel van pHi na acidose, regulatie van celvolume via Na+-influx, en steigerbouw van lamellipodia-uitstulpingen via ERM-actine-interacties mogelijk. Hormonale en groeifactor-signalering kan C-terminale serines (bijv. S703, Thr653) fosforyleren via NHERF1-, ERK- en PKA-routes, waardoor het pHi-setpoint verschuift naar alkaliteit. Pathologisch, bij ischemie-reperfusieletsel, bevordert zuur-geactiveerd NHE1 cytotoxische Na+/Ca2+-overbelasting via reverse-mode NCX-activiteit, wat myocardinfarct verergert. Chronische NHE1-upregulatie is ook betrokken bij tumorinvasie via pericellulaire alkalisering en matrixremodellering, evenals bij harthypertrofie.

Gebruiksinformatie

Toepassing

WB

Verdunning

WB

1:500

Reactiviteit

Mouse, Human, Amphibian, Fish, Avian, Vertebrates

Bron

Mouse Monoclonal Antibody

MW

~100-110 kDa

Opslagbuffer

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

Opslag
(Vanaf de datum van ontvangst)

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

Experimentele Methoden
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.

Experimentele Methoden

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.

Referenties

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

Toepassingsgegevens

WB

Gevalideerd door Selleck

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