Datasheet

Biologische Beschrijving

Specificiteit	NOX2/gp91phox Antibody [G21P21] herkent endogene niveaus van totaal NOX2/gp91phox-eiwit.
Achtergrond	NOX2, ook bekend als gp91phox, is een membraangebonden enzym, het prototypische lid van de NADPH oxidase NOX-superfamilie en produceert superoxide (O2•−), een belangrijke reactieve zuurstofsoort (ROS) die essentieel is voor de aangeboren en adaptieve immuniteit. Het dient als de katalytische kern van het NADPH-oxidasecomplex, voornamelijk te vinden in fagocytische cellen zoals neutrofielen en macrofagen. Structureel vormt NOX2 een heterodimere transmembraankern met p22phox en vereist vier cytosolische subeenheden – p47phox, p67phox, p40phox en Rac1 of Rac2 – voor activering. Bij assemblage katalyseert NOX2 de overdracht van elektronen van NADPH naar moleculaire zuurstof via FAD- en heemcofactoren, waardoor superoxide (O2•−), een reactieve zuurstofsoort (ROS), wordt gegenereerd. Functioneel is NOX2 essentieel voor de respiratoire burst tijdens fagocytose, helpt het bij de vernietiging van pathogenen en speelt het een cruciale rol in de aangeboren immuniteit. De expressie ervan, beïnvloed door ontstekingssignalen, draagt ook bij aan ROS-gemedieerde celsignalering en heeft implicaties in inflammatoire en vasculaire pathologieën. Mutaties in NOX2 kunnen leiden tot chronische granulomateuze ziekte, gekenmerkt door een verhoogde gevoeligheid voor infecties.

Specificiteit

NOX2/gp91phox Antibody [G21P21] herkent endogene niveaus van totaal NOX2/gp91phox-eiwit.

Achtergrond

NOX2, ook bekend als gp91phox, is een membraangebonden enzym, het prototypische lid van de NADPH oxidase NOX-superfamilie en produceert superoxide (O2•−), een belangrijke reactieve zuurstofsoort (ROS) die essentieel is voor de aangeboren en adaptieve immuniteit. Het dient als de katalytische kern van het NADPH-oxidasecomplex, voornamelijk te vinden in fagocytische cellen zoals neutrofielen en macrofagen. Structureel vormt NOX2 een heterodimere transmembraankern met p22phox en vereist vier cytosolische subeenheden – p47phox, p67phox, p40phox en Rac1 of Rac2 – voor activering. Bij assemblage katalyseert NOX2 de overdracht van elektronen van NADPH naar moleculaire zuurstof via FAD- en heemcofactoren, waardoor superoxide (O2•−), een reactieve zuurstofsoort (ROS), wordt gegenereerd. Functioneel is NOX2 essentieel voor de respiratoire burst tijdens fagocytose, helpt het bij de vernietiging van pathogenen en speelt het een cruciale rol in de aangeboren immuniteit. De expressie ervan, beïnvloed door ontstekingssignalen, draagt ook bij aan ROS-gemedieerde celsignalering en heeft implicaties in inflammatoire en vasculaire pathologieën. Mutaties in NOX2 kunnen leiden tot chronische granulomateuze ziekte, gekenmerkt door een verhoogde gevoeligheid voor infecties.

Gebruiksinformatie

Toepassing

WB

Verdunning

WB

1:5000

Reactiviteit

Human, Mouse, Rat

Bron

Rabbit Monoclonal Antibody

MW

60 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 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. 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 1:5000), 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 1347. 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)

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 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

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 1:5000), 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

1347. 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)

Referenties

https://pubmed.ncbi.nlm.nih.gov/36241643/

Toepassingsgegevens

WB

Gevalideerd door Selleck

Lane 1: MCF-7
Lane 2: HepG2
Lane 3: Mouse brain
Lane 4: Rat brain