DUSP6/MKP3 Antibody [E9K23] | Primary Antibodies

Application: Reactivity: Human

Lane 1: NCI-H3255, Lane 2: DLD-1

Usage Information

Dilution
WB1:1000 IP1:200

Application
WB, IP

Reactivity
Human

Source
Rabbit 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
42 kDa

Positive Control	NCI-H3255 cellS; NCI-H441 cells; DLD-1 cells
Negative Control	HeLa cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Tris-HCL 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 wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Tris-HCL 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. Lyse the cells by adding an appropriate volume of RIPA/Tris-HCL 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 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/Tris-HCL 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 wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Tris-HCL 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. Lyse the cells by adding an appropriate volume of RIPA/Tris-HCL 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

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
DUSP6/MKP3 Antibody [E9K23] detects endogenous levels of total DUSP6/MKP3 protein.

Subcellular Location
Cytoplasm

Uniprot ID
Q16828

Clone
E9K23

Synonym(s)
Dual specificity protein phosphatase 6; Mitogen-activated protein kinase phosphatase 3; MKP-3; DUSP6

Background
Dual‑specificity phosphatase 6 (DUSP6), also known as MAP kinase phosphatase‑3 (MKP3/Pyst1), is a cytoplasmic dual‑specificity phosphatase that selectively dephosphorylates and inactivates ERK1/2 within the classical MAPK cascade, thereby shaping the duration, intensity, and spatial profile of ERK signaling. DUSP6 contains an N‑terminal kinase‑interaction motif (KIM) that docks to the common docking site of ERK2, positioning the C‑terminal catalytic phosphatase domain to remove phosphate groups from both Thr and Tyr residues within the ERK activation‑loop heptapeptide (Thr‑Glu‑Tyr), while structural studies show that the phosphatase domain forms a shallow, active pocket that accommodates the doubly phosphorylated ERK activation segment and permits ERK‑stimulated phosphatase activity. ERK‑dependent transcriptional induction of DUSP6 creates a negative feedback loop that limits ERK amplitude and duration, and genetic loss of Dusp6/Mkp3 in mouse models enhances ERK activation and underlies developmental defects such as skeletal dwarfism and craniosynostosis, phenotypes that mirror hyperactive FGFR signaling, demonstrating its role as an intrinsic brake on FGF/ERK outputs. DUSP6 expression is both ERK‑regulated and oncogene‑tuned: DUSP6 loss or downregulation heightens ERK activity and promotes proliferation and tumorigenesis in epithelial cancers, whereas DUSP6 overexpression in some settings, including ovarian and breast carcinoma lines, dampens ERK signaling, lowers cyclin D3 levels, increases the G0/G1 ratio, and can either sensitize cells to chemotherapeutic apoptosis or, conversely, promote quiescence‑associated therapy resistance.

Background

Dual‑specificity phosphatase 6 (DUSP6), also known as MAP kinase phosphatase‑3 (MKP3/Pyst1), is a cytoplasmic dual‑specificity phosphatase that selectively dephosphorylates and inactivates ERK1/2 within the classical MAPK cascade, thereby shaping the duration, intensity, and spatial profile of ERK signaling. DUSP6 contains an N‑terminal kinase‑interaction motif (KIM) that docks to the common docking site of ERK2, positioning the C‑terminal catalytic phosphatase domain to remove phosphate groups from both Thr and Tyr residues within the ERK activation‑loop heptapeptide (Thr‑Glu‑Tyr), while structural studies show that the phosphatase domain forms a shallow, active pocket that accommodates the doubly phosphorylated ERK activation segment and permits ERK‑stimulated phosphatase activity. ERK‑dependent transcriptional induction of DUSP6 creates a negative feedback loop that limits ERK amplitude and duration, and genetic loss of Dusp6/Mkp3 in mouse models enhances ERK activation and underlies developmental defects such as skeletal dwarfism and craniosynostosis, phenotypes that mirror hyperactive FGFR signaling, demonstrating its role as an intrinsic brake on FGF/ERK outputs. DUSP6 expression is both ERK‑regulated and oncogene‑tuned: DUSP6 loss or downregulation heightens ERK activity and promotes proliferation and tumorigenesis in epithelial cancers, whereas DUSP6 overexpression in some settings, including ovarian and breast carcinoma lines, dampens ERK signaling, lowers cyclin D3 levels, increases the G0/G1 ratio, and can either sensitize cells to chemotherapeutic apoptosis or, conversely, promote quiescence‑associated therapy resistance.

References
https://pubmed.ncbi.nlm.nih.gov/22812510/ https://pubmed.ncbi.nlm.nih.gov/17164422/

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

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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%