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Phospho-STAT6 (Tyr641) Antibody [G7E12]

Cat.No.: F4042

Application: Reactivity: Mouse, Rat, Human

Lane 1: Hela (serum starvation, 24 h), Lane 2: Hela (serum starvation, 24 h; IL-4, 100 ng/ml, 15 min)

Usage Information

Dilution
WB1:1000 IP1:30 FCM1:500

Application
WB, IP, FCM

Reactivity
Mouse, Rat, 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 Observed MW
94 kDa 110 kDa, 94 kDa, 75 kDa, 81 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	Mouse ileum tissue; RAW264.7 cell (serum starvation, 24 h; mIL-4, 100 ng/ml, 15 min); 2.4G2 cell (serum starvation, 24 h; rIL-4, 100 ng/ml, 15 min); Daudi cell (serum starvation, 24 h; IL-4, 100 ng/ml, 15 min)
Negative Control	RAW264.7cell (serum starved for 24 hours); 2.4G2 cell (serum starved for 24 hours); Daudi cell (serum starved for 24 hours)

Exprimental 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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.

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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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
Phospho-STAT6 (Tyr641) Antibody [G7E12] detects endogenous levels of total STAT6 protein only when it is phosphorylated at Tyr641.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
P42226

Clone
G7E12

Synonym(s)
Signal transducer and transcription activator 6; Stat6

Background
Phospho-STAT6 (Tyr641) is the activated form of Signal Transducer and Activator of Transcription 6 (STAT6), a critical signaling protein belonging to the STAT family involved in cytokine-mediated cellular responses. STAT6 is composed of several domains, including an N-terminal coiled-coil domain, a DNA-binding domain, an SH2 domain essential for receptor binding and dimerization, a linker domain, and a C-terminal transactivation domain. Phosphorylation at Tyr641, catalyzed primarily by Janus kinases (JAKs) upon cytokine stimulation like interleukin-4 (IL-4) and interleukin-13 (IL-13), induces STAT6 dimerization through reciprocal SH2-phosphotyrosine interactions. This active dimer translocates to the nucleus to regulate transcription of target genes associated with T-helper cell differentiation and type 2 immune responses. The phosphorylated Tyr641 tail forms intermolecular gates with SH2 domains of adjacent STAT6 monomers, stabilizing the DNA-bound dimer complex. Additional modulation of STAT6 activity occurs via interactions with other proteins, such as STAT2 during IFN-α signaling, and protein phosphatase 2A fine-tunes the IL-4 mediated signaling cascade.

Background

Phospho-STAT6 (Tyr641) is the activated form of Signal Transducer and Activator of Transcription 6 (STAT6), a critical signaling protein belonging to the STAT family involved in cytokine-mediated cellular responses. STAT6 is composed of several domains, including an N-terminal coiled-coil domain, a DNA-binding domain, an SH2 domain essential for receptor binding and dimerization, a linker domain, and a C-terminal transactivation domain. Phosphorylation at Tyr641, catalyzed primarily by Janus kinases (JAKs) upon cytokine stimulation like interleukin-4 (IL-4) and interleukin-13 (IL-13), induces STAT6 dimerization through reciprocal SH2-phosphotyrosine interactions. This active dimer translocates to the nucleus to regulate transcription of target genes associated with T-helper cell differentiation and type 2 immune responses. The phosphorylated Tyr641 tail forms intermolecular gates with SH2 domains of adjacent STAT6 monomers, stabilizing the DNA-bound dimer complex. Additional modulation of STAT6 activity occurs via interactions with other proteins, such as STAT2 during IFN-α signaling, and protein phosphatase 2A fine-tunes the IL-4 mediated signaling cascade.

References
https://pubmed.ncbi.nlm.nih.gov/21442426/ https://pubmed.ncbi.nlm.nih.gov/26506089/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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%