Fiche technique

Description biologique

Spécificité	NAE1/APPBP1 Antibody [N23N11] reconnaît les niveaux endogènes de la protéine NAE1/APPBP1 totale. Cet anticorps ne réagit pas de manière croisée avec les enzymes E1 activating pour Ubiquitin ou d'autres protéines de type ubiquitine.
Contexte	NAE1 (également connu sous le nom d'APPBP1) est un composant clé du complexe enzymatique E1 activant le NEDD8, qui est crucial pour la neddylation – un processus qui implique l'attachement de NEDD8 à des protéines cibles via une voie séquentielle en trois étapes facilitée par les enzymes E1, E2 et E3. NAE1, d'un poids moléculaire d'environ 60 kDa, s'associe à UBA3, qui agit comme sous-unité catalytique, tandis qu'APPBP1 accélère la cinétique de l'étape d'activation. La neddylation, régulée par ce complexe, est impliquée dans diverses voies cellulaires et dans la physiopathologie de plusieurs maladies. Les mutations de NAE1 peuvent entraîner une diminution de l'abondance de NAE1 et sont associées à des phénotypes cliniques tels que la perte neuronale, l'aggravation de la lymphopénie lors d'infections et des défauts de développement squelettique. NAE1 est essentiel à l'homéostasie cellulaire et à la protection contre la mort cellulaire dans des conditions de stress, telles que les infections. NAE1 joue un rôle majeur dans le développement et la réponse au stress cellulaire, soulignant son importance dans le maintien de la fonction cellulaire.

Spécificité

NAE1/APPBP1 Antibody [N23N11] reconnaît les niveaux endogènes de la protéine NAE1/APPBP1 totale. Cet anticorps ne réagit pas de manière croisée avec les enzymes E1 activating pour Ubiquitin ou d'autres protéines de type ubiquitine.

Contexte

NAE1 (également connu sous le nom d'APPBP1) est un composant clé du complexe enzymatique E1 activant le NEDD8, qui est crucial pour la neddylation – un processus qui implique l'attachement de NEDD8 à des protéines cibles via une voie séquentielle en trois étapes facilitée par les enzymes E1, E2 et E3. NAE1, d'un poids moléculaire d'environ 60 kDa, s'associe à UBA3, qui agit comme sous-unité catalytique, tandis qu'APPBP1 accélère la cinétique de l'étape d'activation. La neddylation, régulée par ce complexe, est impliquée dans diverses voies cellulaires et dans la physiopathologie de plusieurs maladies. Les mutations de NAE1 peuvent entraîner une diminution de l'abondance de NAE1 et sont associées à des phénotypes cliniques tels que la perte neuronale, l'aggravation de la lymphopénie lors d'infections et des défauts de développement squelettique. NAE1 est essentiel à l'homéostasie cellulaire et à la protection contre la mort cellulaire dans des conditions de stress, telles que les infections. NAE1 joue un rôle majeur dans le développement et la réponse au stress cellulaire, soulignant son importance dans le maintien de la fonction cellulaire.

Informations dutilisation

Application

WB

Dilution

WB

1:1000

Réactivité

Human, Mouse, Monkey

Source

Rabbit Monoclonal Antibody

MW

60 kDa

Tampon de stockage

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

Stockage
(À partir de la date de réception)

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

Méthodes expérimentales
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 852. 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.

Méthodes expérimentales

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

852. 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.

Références

https://pubmed.ncbi.nlm.nih.gov/36608681/

Données dapplication

WB

Validé par Selleck

Lane 1: 293T
Lane 2: COS-7
Lane 3: U-2 OS