Datasheet

Biological Description

Specificity	ID3 Rabbit mAb recognizes endogenous levels of total ID3 protein.
Background	ID3 (Inhibitor of DNA-binding 3) is a member of the helix-loop-helix (HLH) family of transcription factors, playing a central role in regulating key cellular processes such as differentiation, proliferation, and DNA damage response (DDR). Unlike other HLH proteins, ID3 lacks a basic DNA-binding domain and instead functions by binding to and sequestering E-proteins, transcription factors that promote cellular differentiation. This interaction prevents E-proteins from activating genes associated with differentiation, thus maintaining a progenitor or undifferentiated state. ID3's structure comprises a basic HLH domain, critical for protein-protein interactions but not for direct DNA binding. Upon DNA damage, ID3 undergoes phosphorylation at serine 65 by the ATM (Ataxia Telangiectasia Mutated) kinase, a key regulator of the DDR. This phosphorylation event is essential for enhancing its interactions with proteins involved in DNA repair, particularly those involved in the repair of double-strand breaks (DSBs), such as the MRN complex (NBS1, RAD50, MRE11), MDC1, and RECQL. These interactions are crucial for facilitating repair through homologous recombination (HR), where ID3 promotes the recruitment of key repair enzymes, such as RAD51, and facilitates DNA end resection. It also helps in transcriptional regulation, where it influences the expression of DNA repair genes, such as those involved in HR and Fanconi Anemia (FA) pathways, through its interactions with transcription factors like E2F1 and chromatin remodelling factors like PRMT5. Loss of ID3 results in impaired DNA repair and increased sensitivity to DNA-damaging agents, including ionizing radiation and chemotherapeutic agents like cisplatin. ID3 has a dual role in cancer biology, where it can act both as a tumor suppressor and as a promoter, depending on the context. In some cancers, such as hematological malignancies and colorectal cancer, ID3 promotes cancer stem cell self-renewal and chemoresistance, while in others, it suppresses tumor progression by promoting differentiation and preventing uncontrolled cell proliferation.

Specificity

ID3 Rabbit mAb recognizes endogenous levels of total ID3 protein.

Background

ID3 (Inhibitor of DNA-binding 3) is a member of the helix-loop-helix (HLH) family of transcription factors, playing a central role in regulating key cellular processes such as differentiation, proliferation, and DNA damage response (DDR). Unlike other HLH proteins, ID3 lacks a basic DNA-binding domain and instead functions by binding to and sequestering E-proteins, transcription factors that promote cellular differentiation. This interaction prevents E-proteins from activating genes associated with differentiation, thus maintaining a progenitor or undifferentiated state. ID3's structure comprises a basic HLH domain, critical for protein-protein interactions but not for direct DNA binding. Upon DNA damage, ID3 undergoes phosphorylation at serine 65 by the ATM (Ataxia Telangiectasia Mutated) kinase, a key regulator of the DDR. This phosphorylation event is essential for enhancing its interactions with proteins involved in DNA repair, particularly those involved in the repair of double-strand breaks (DSBs), such as the MRN complex (NBS1, RAD50, MRE11), MDC1, and RECQL. These interactions are crucial for facilitating repair through homologous recombination (HR), where ID3 promotes the recruitment of key repair enzymes, such as RAD51, and facilitates DNA end resection. It also helps in transcriptional regulation, where it influences the expression of DNA repair genes, such as those involved in HR and Fanconi Anemia (FA) pathways, through its interactions with transcription factors like E2F1 and chromatin remodelling factors like PRMT5. Loss of ID3 results in impaired DNA repair and increased sensitivity to DNA-damaging agents, including ionizing radiation and chemotherapeutic agents like cisplatin. ID3 has a dual role in cancer biology, where it can act both as a tumor suppressor and as a promoter, depending on the context. In some cancers, such as hematological malignancies and colorectal cancer, ID3 promotes cancer stem cell self-renewal and chemoresistance, while in others, it suppresses tumor progression by promoting differentiation and preventing uncontrolled cell proliferation.

Usage Information

Application

WB, IP, IF, FCM

Dilution

WB	IP	IF	FCM
1:1000	1:200	1:400 - 1:1600	1:400 - 1:1600

Reactivity

Human

Source

Rabbit

MW

13 KDa

Storage Buffer

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

Storage
(from the date of receipt)

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

Exprimental Methods
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.

References

Application Data

WB

Validated by Selleck

Lane 1: Hela
Lane 2: Jurkat
Lane 3: Ramos