ID3 Antibody [J14L22]

N° de catalogue F2652

Imprimer

Description biologique

Spécificité

ID3 Antibody [J14L22] reconnaît les niveaux endogènes de la protéine ID3 totale.
Contexte

ID3 (Inhibitor of DNA-binding 3) est un membre de la famille des facteurs de transcription hélice-boucle-hélice (HLH), jouant un rôle central dans la régulation de processus cellulaires clés tels que la différenciation, la prolifération et la réponse aux DNA Damage/DNA Repair (DDR). Contrairement à d'autres protéines HLH, ID3 ne possède pas de domaine de liaison à l'ADN basique et fonctionne plutôt en se liant et en séquestrant les protéines E, des facteurs de transcription qui favorisent la différenciation cellulaire. Cette interaction empêche les protéines E d'activer les gènes associés à la différenciation, maintenant ainsi un état de progéniteur ou non différencié. La structure d'ID3 comprend un domaine HLH basique, essentiel pour les interactions protéine-protéine mais pas pour la liaison directe à l'ADN. Lors de DNA Damage/DNA Repair, ID3 subit une phosphorylation à la sérine 65 par la kinase ATM (Ataxia Telangiectasia Mutated), un régulateur clé de la DDR. Cet événement de phosphorylation est essentiel pour améliorer ses interactions avec les protéines impliquées dans la DNA Damage/DNA Repair, en particulier celles impliquées dans la réparation des cassures double brin (DSB), telles que le complexe MRN (NBS1, RAD50, MRE11), MDC1 et RECQL. Ces interactions sont cruciales pour faciliter la réparation par recombinaison homologue (HR), où ID3 favorise le recrutement d'enzymes de réparation clés, telles que RAD51, et facilite la résection des extrémités de l'ADN. Il aide également à la régulation transcriptionnelle, où il influence l'expression des gènes de DNA Damage/DNA Repair, tels que ceux impliqués dans les voies HR et Fanconi Anemia (FA), par ses interactions avec des facteurs de transcription comme E2F1 et des facteurs de remodelage de la chromatine comme PRMT5. La perte d'ID3 entraîne une réparation de l'ADN altérée et une sensibilité accrue aux agents endommageant l'ADN, y compris les rayonnements ionisants et les agents chimiothérapeutiques comme le cisplatine. ID3 a un double rôle en biologie du cancer, où il peut agir à la fois comme suppresseur de tumeur et comme promoteur, selon le contexte. Dans certains cancers, tels que les hémopathies malignes et le cancer colorectal, ID3 favorise l'auto-renouvellement des cellules souches cancéreuses et la chimiorésistance, tandis que dans d'autres, il supprime la progression tumorale en favorisant la différenciation et en empêchant la prolifération cellulaire incontrôlée.

Informations dutilisation

Application WB, IP, IF, FCM Dilution
WB IP IF FCM
1:1000 1:200 1:400 - 1:1600 1:400 - 1:1600
Réactivité Human
Source Rabbit Monoclonal Antibody MW 13 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
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 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/Nuclear 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/Nuclear 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: 20%. 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications
Recommended conditions for wet transfer: 200 mA, 60 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
1223. 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 300s is recommended)
IF
Experimental Protocol:
 
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
 
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
 
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
Wash the sample with PBS for 3 times, 3 minutes each time.
 
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
 
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
 
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
 
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.

Références

  • https://pubmed.ncbi.nlm.nih.gov/34718742/
  • https://pubmed.ncbi.nlm.nih.gov/28122577/

Données dapplication

WB

Validé par Selleck

  • F2652-wb
    Lane 1: Hela
    Lane 2: Jurkat
    Lane 3: Ramos

IF

Validé par Selleck

  • F2652-IF
    Immunofluorescent analysis of Hela cells using F2652 (green, 1:400), Hoechst (blue) and tubulin (Red).