Description
Human SHP2 (Y542) Phosphorylation ELISA Kit (BA0255) (BA0255)
The Human SHP2 (Y542) Phosphorylation ELISA Kit (SKU: BA0255) provides a rapid and sensitive fluorimetric cell-based assay for measuring the phosphorylated protein pSHP2(Y542) in human cells. Src Homology Region 2 Domain-containing Phosphatase-2 (SHP2, also known as SHPTP2, PTPN11 or PTP2C) is a widely expressed protein-tyrosine phosphatase that plays regulatory roles in mitogenic activation, metabolic control, transcription regulation and cell migration. The assay normalises the level of phosphorylation to the total protein content in the same well, greatly minimising well-to-well variation. Cells cultured directly in a 96-well plate are fixed and permeabilised, and the phosphorylation of SHP2(Y542) is measured using a specific primary antibody followed by an HRP-conjugated secondary antibody with a fluorogenic substrate, alongside a fluorescent reagent that measures total protein. This simple and efficient format eliminates the need for cell lysate preparation and is well suited to studying signalling pathways and the effects of inhibitors, siRNA or activators. Total assay time has been reduced from the standard 21 hours to just 6.5 hours.
| Product Name: | Human SHP2 (Y542) Phosphorylation ELISA Kit (BA0255) |
| SKU: | BA0255 |
| Detection Method: | Cell-Based ELISA (fluorimetric, FL 530/585 nm and 360/450 nm) |
| Sample Type: | ['Cells'] |
| Species Reactivity: | Human |
| Assay Time: | 6.5 hours total (hands-on time 2.5 hours) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months |
| Shipping: | Gel Pack |
Fluorimetric cell-based assay for the quantitative determination of SHP2(Y542) phosphorylation status in whole human cells, with normalisation to total cellular protein in the same well.
- New and improved: total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hours).
- Simple and convenient: cells are cultured directly in 96-well plates with no cell lysis necessary.
- Accurate and high-throughput: protein phosphorylation is normalised to total cellular protein in the same well, greatly minimising well-to-well variation and enabling automation for thousands of samples per day.
- Determination of SHP2(Y542) phosphorylation status in whole cells.
- Evaluation of pathway modulation by activators, inhibitors, siRNA and similar agents.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Prepare 1x Wash Buffer by diluting Stock Wash Buffer 40-fold with dH2O (e.g. 10 mL Stock Wash Buffer + 390 mL dH2O); reserve 6 mL 1x Wash Buffer for the Detection step. Assay samples in triplicate or more, and include a Protein Blank (no cells) and a Sample Blank (cells with no Ab1, only Ab2). |
| 2 | Culture and treat cells: seed 100 µL of 1-3×10^4 adherent cells (or 4-10×10^4 suspension cells) into each well of a black 96-well culture plate, add 100 µL cell-free media to three wells for the Protein Blank, and incubate overnight at 37°C. Treat cells as desired. |
| 3 | Fix cells with freshly prepared formaldehyde solution (4 wt% for adherent cells; 8 wt% for suspension cells) for 20 min at room temperature. Remove and wash twice with 200 µL 1x Wash Buffer (3 min gentle shaking per wash). |
| 4 | Quench with 100 µL Quench Buffer (2.2 mL of 3% H2O2 + 8.8 mL 1x Wash Buffer) for 20 min at room temperature, then wash three times with 200 µL 1x Wash Buffer. |
| 5 | Block with 100 µL Blocking Buffer for 1 hr at room temperature. |
| 6 | Add primary antibody: prepare Ab1 Mixture (Ab1 in Blocking Buffer at 1:1000). Add 50 µL Blocking Buffer to Sample Blank wells and 50 µL Ab1 Mixture to Sample wells; incubate 90 min at room temperature (or overnight at 2-8°C) with gentle shaking, then wash three times. |
| 7 | Add secondary antibody: prepare Ab2 Mixture (Ab2 in Blocking Buffer at 1:1000). Add 50 µL to all wells; incubate 90 min at room temperature with gentle shaking, then wash four times. |
| 8 | Detection: prepare HRP Substrate (60 µL Dye Reagent + 6 mL 1x Wash Buffer + 6 µL 3% H2O2). Add 50 µL to each well and incubate 30 min at room temperature in the dark. |
| 9 | Add 50 µL Protein Stain to each well and incubate a further 5 min at room temperature in the dark. |
| 10 | Read the plate at λex/em = 530/585 nm for pSHP2(Y542) and at λex/em = 360/450 nm for total protein. |
Calculate the mean pTarget fluorescence at 530/585 nm for the Sample Blank (FpTarget.Blank) and Sample wells (FpTarget.Sample), and the mean protein fluorescence at 360/450 nm for the Protein Blank (FProtein.Blank) and Sample wells (FProtein.Sample). Determine specific values: ΔFpTarget = FpTarget.Sample – FpTarget.Blank and ΔFProtein = FProtein.Sample – FProtein.Blank. Normalised pTarget = (ΔFpTarget / ΔFProtein) / (ΔFpTarget / ΔFProtein)o, where the denominator is the control reference value (e.g. time zero in kinetic studies or untreated wells in drug potency studies).
| Component | Quantity | Storage |
| Stock Wash Buffer | 25 mL | -20°C |
| Blocking Buffer | 25 mL | -20°C |
| Protein Stain | 6 mL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Ab1 | 10 µL | -20°C |
| Ab2 (gM-HRP) | 10 µL | -20°C |
