Fiche technique

Description biologique

Spécificité	PAN TEAD Antibody [A12P9] détecte les niveaux endogènes de la protéine Pan-TEAD totale.
Contexte	La famille TEAD de facteurs de transcription, comprenant TEAD1, TEAD2, TEAD3 et TEAD4, est un groupe conservé de protéines liant l'ADN qui régulent les gènes contrôlant la prolifération cellulaire, la différenciation, la survie, l'apoptose, la croissance tissulaire et la régénération. Ils agissent comme des effecteurs centraux en aval de la voie de signalisation Hippo, intégrant des entrées mécaniques et biochimiques pour maintenir l'homéostasie tissulaire et contrôler la taille des organes. Toutes les protéines TEAD partagent un domaine de liaison à l'ADN TEA N-terminal (~70 à 75 acides aminés) avec un repliement hélice-boucle-hélice qui reconnaît les éléments MCAT dans les promoteurs cibles, et un domaine de liaison à YAP (YBD) C-terminal avec un repliement de type immunoglobuline qui recrute les coactivateurs transcriptionnels YAP et TAZ. Ces coactivateurs sont essentiels pour une activation transcriptionnelle robuste des gènes cibles tels que CTGF et CYR61, car les TEAD seuls ont une faible activité intrinsèque. La fonction TEAD coordonnée influence la dynamique cytosquelettique, la cicatrisation des plaies et la régénération tissulaire. La dérégulation de tout membre de la famille TEAD, souvent par une activation aberrante de YAP/TAZ, entraîne des programmes transcriptionnels oncogènes, favorise les métastases et est associée à des troubles du développement, des maladies fibroprolifératives et des cardiomyopathies.

Spécificité

PAN TEAD Antibody [A12P9] détecte les niveaux endogènes de la protéine Pan-TEAD totale.

Contexte

La famille TEAD de facteurs de transcription, comprenant TEAD1, TEAD2, TEAD3 et TEAD4, est un groupe conservé de protéines liant l'ADN qui régulent les gènes contrôlant la prolifération cellulaire, la différenciation, la survie, l'apoptose, la croissance tissulaire et la régénération. Ils agissent comme des effecteurs centraux en aval de la voie de signalisation Hippo, intégrant des entrées mécaniques et biochimiques pour maintenir l'homéostasie tissulaire et contrôler la taille des organes. Toutes les protéines TEAD partagent un domaine de liaison à l'ADN TEA N-terminal (~70 à 75 acides aminés) avec un repliement hélice-boucle-hélice qui reconnaît les éléments MCAT dans les promoteurs cibles, et un domaine de liaison à YAP (YBD) C-terminal avec un repliement de type immunoglobuline qui recrute les coactivateurs transcriptionnels YAP et TAZ. Ces coactivateurs sont essentiels pour une activation transcriptionnelle robuste des gènes cibles tels que CTGF et CYR61, car les TEAD seuls ont une faible activité intrinsèque. La fonction TEAD coordonnée influence la dynamique cytosquelettique, la cicatrisation des plaies et la régénération tissulaire. La dérégulation de tout membre de la famille TEAD, souvent par une activation aberrante de YAP/TAZ, entraîne des programmes transcriptionnels oncogènes, favorise les métastases et est associée à des troubles du développement, des maladies fibroprolifératives et des cardiomyopathies.

Informations dutilisation

Application

WB

Dilution

WB

1:1000

Réactivité

Human

Source

Rabbit Monoclonal Antibody

MW

48 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/Nuclear 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/Nuclear 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/Nuclear 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 1:1000), 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 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.

Méthodes expérimentales

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear 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/Nuclear 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/Nuclear 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 1:1000), 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

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.

Références

https://pubmed.ncbi.nlm.nih.gov/18579750/
https://pubmed.ncbi.nlm.nih.gov/20123908/

Données dapplication

WB

Validé par Selleck

Lane 1: HeLa, Lane 2: SW480