phospho-Tyrosine Monoclonal Antibody (pY20), PerCP-eFluor™ 710, eBioscience™, Invitrogen™

Description

The role of tyrosine phosphorylation in transduction of the mitogenic signal from transmembrane receptors and in transformation by oncogene tyrosine kinases has been the subject of intense investigation for several years. While the phosphorylation of specific tyrosine residues has been shown to be a primary mechanism of signal transduction during normal mitogenesis, cell cycle progression and oncogenic transformation, its role in other areas such as differentiation and gap junction communication, is a matter of active and ongoing research. Antibodies that specifically recognize phosphorylated tyrosine residues have proved to be invaluable to the study of tyrosine -phosphorylated proteins and the biochemical pathways in which they function. The fluorescein (FITC) conjugate of clone PY20 anti-phosphotyrosine is especially useful for the detection of these P-Tyr proteins in immunohistochemical and immunocytochemical protocols in situations wherein the use of a secondary antibody would complicate detection of the protein(s) of interest.

Description: The pY20 monoclonal antibody recognizes phosphorylated tyrosine residues (p-Tyr) on proteins. Numerous intracellular signaling cascades are propagated via phosphorylation of specific tyrosine on signaling proteins. The detection of p-Tyr residues is valuable for the characterization and purification of phosphorylated proteins and the biochemical pathways that they are involved in.Applications Reported: This pY20 antibody has been reported for use in intracellular staining followed by flow cytometric analysis.Applications Tested: This pY20 antibody has been pre-titrated and tested by intracellular staining of normal mouse lymph node cells. This can be used at 5 μL (1.0 μg) per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Staining Protocol: All protocols work well for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Use of Protocol B: One-step protocol: intracellular (nuclear) proteins is recommended for staining of transcription factors in conjunction with surface and phosphorylated intracellular (cytoplasmic) protein(s). Protocol C: Two-step protocol: Fixation/Methanol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to Clone Performance Following Fixation/Permeabilization located in the Best Protocols Section under the Resources tab online). All Protocols can be found in the Flow Cytometry Protocols: Staining Intracellular Antigens for Flow Cytometry Protocol located in the Best Protocols Section under the Resources tab online.PerCP-eFluor™ 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100 μL of cell sample + 100 μL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. PerCP-eFluor™ 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100 μL of cell sample + 100 μL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.Filtration: 0.2μm post-manufacturing filtered.