|
Toll Free: (877) 796-6397 -- USA and Canada only -- |
Fax: +1-832-582-8590 Orders: +1-832-582-8158 |
Tech Support: +1-832-582-8158 Ext:3 Please provide your Order Number in the email. |
Biological Description
| Specificity | Phospho-SHP-2 (Tyr 580) Rabbit mAb detects endogenous level of SHP2 only when phosphorylated at Tyr580. |
|---|---|
| Background | Phospho-SHP-2 (Tyr 580), encoded by the PTPN11 gene, is a cytoplasmic protein tyrosine phosphatase that plays a crucial role in growth factor and cytokine signaling. Structurally, SHP-2 contains two SH2 domains (N-SH2 and C-SH2) and a catalytic PTP domain, where the N-SH2 domain maintains an autoinhibited conformation that is relieved upon binding to phosphotyrosine residues. SHP-2 is ubiquitously expressed and functions in diverse cellular processes including proliferation, survival, adhesion, migration, and differentiation. It acts downstream of various receptors, including PDGFR and EGFR, primarily by facilitating Ras/ERK pathway activation through interactions with adaptor proteins like Gab1. SHP-2 is phosphorylated at several tyrosine residues—most notably Y542 and Y580 in the C-terminal region, which serve as docking sites for Grb2/SOS and may modulate catalytic activity and signal propagation. Endogenous Abl kinases were identified as the elusive kinases that phosphorylate SHP-2 on Y580, enhancing or modulating its role in ERK pathway activation and cell cycle progression. SHP-2 phosphorylation is essential for sustained ERK signaling and mitogenic responses to growth factors such as PDGF and EGF, highlighting its importance in both physiological growth and oncogenic transformation, particularly in BCR-Abl-driven leukemogenesis. |
Usage Information
| Application | WB, IP, FCM | Dilution |
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| Reactivity | Mouse, Rat | ||||||||
| Source | Rabbit | MW | 72 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 | ||||||
| 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 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/NP-40 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/NP-40 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
1389. 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 180s is recommended)
|