DEC1 Antibody [D7J6] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: HepG2

Usage Information

Dilution
WB1:100-1:1000 IP1:100-1:200

Application
WB, IP, ELISA

Reactivity
Human, Mouse, Rat

Source
Mouse Monoclonal Antibody

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

Predicted MW
43 kDa

Positive Control	Hep G2 cells; 293 cells
Negative Control

Experimental Methods

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. 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/NP-40 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/NP-40 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.

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.

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/NP-40 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/NP-40 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.

Datasheet & SDS

Biological Description

Specificity
DEC1 Antibody [D7J6] detects endogenous levels of total DEC1 protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
O14503

Clone
D7J6

Synonym(s)
Class E basic helix-loop-helix protein 40; bHLHe40; Class B basic helix-loop-helix protein 2 (bHLHb2); DEC1; Enhancer-of-split and hairy-related protein 2 (SHARP-2); BHLHE40; BHLHB2; DEC1; SHARP2; STRA13

Background
DEC1 (BHLHE40) belongs to the basic helix-loop-helix family of transcription factors alongside DEC2, characterized by a DNA-binding basic domain and helix-loop-helix dimerization motif that recognizes E-box sequences (CACGTG) in target gene promoters to modulate circadian rhythm, differentiation, and stress responses. DEC1 organizes an N-terminal transactivation domain, central Orange domain for heterodimer selectivity, and nuclear localization signals flanking the bHLH region that confers specificity for class C E-boxes while repressing CLOCK-BMAL1 heterodimers essential for circadian oscillation. DEC1 transcriptionally represses pro-apoptotic genes under serum deprivation by stabilizing cyclin E and inhibiting caspase activation through selective suppression of mitochondrial pathways, while hypoxia-inducible factor signaling induces DEC1 expression that cooperates with HIF1α to transactivate VEGF and glycolytic enzymes for adaptive metabolic reprogramming. Interaction with p53 modulates DNA damage responses, where DEC1 attenuates MIC-1 induction but enhances survivin to balance survival versus death, alongside direct binding to TGF-β1 promoters that amplifies Smad3 phosphorylation and SNAIL expression driving epithelial-mesenchymal transition through N-cadherin upregulation and E-cadherin repression. DEC1 coordinates chondrocyte maturation and osteoblast differentiation during skeletal development, with rhythmic expression in the suprachiasmatic nucleus maintaining peripheral clock entrainment.

Background

DEC1 (BHLHE40) belongs to the basic helix-loop-helix family of transcription factors alongside DEC2, characterized by a DNA-binding basic domain and helix-loop-helix dimerization motif that recognizes E-box sequences (CACGTG) in target gene promoters to modulate circadian rhythm, differentiation, and stress responses. DEC1 organizes an N-terminal transactivation domain, central Orange domain for heterodimer selectivity, and nuclear localization signals flanking the bHLH region that confers specificity for class C E-boxes while repressing CLOCK-BMAL1 heterodimers essential for circadian oscillation. DEC1 transcriptionally represses pro-apoptotic genes under serum deprivation by stabilizing cyclin E and inhibiting caspase activation through selective suppression of mitochondrial pathways, while hypoxia-inducible factor signaling induces DEC1 expression that cooperates with HIF1α to transactivate VEGF and glycolytic enzymes for adaptive metabolic reprogramming. Interaction with p53 modulates DNA damage responses, where DEC1 attenuates MIC-1 induction but enhances survivin to balance survival versus death, alongside direct binding to TGF-β1 promoters that amplifies Smad3 phosphorylation and SNAIL expression driving epithelial-mesenchymal transition through N-cadherin upregulation and E-cadherin repression. DEC1 coordinates chondrocyte maturation and osteoblast differentiation during skeletal development, with rhythmic expression in the suprachiasmatic nucleus maintaining peripheral clock entrainment.

References
https://pubmed.ncbi.nlm.nih.gov/26819638/ https://pubmed.ncbi.nlm.nih.gov/12119049/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%