Antibody data
- Antibody Data
- Antigen structure
- References [1]
- Comments [0]
- Validations
- Flow cytometry [1]
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- Product number
- 46-9076-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-LCK (Tyr505) Monoclonal Antibody (SRRCHA), PerCP-eFluor™ 710, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Description
- Description: This SRRCHA monoclonal antibody recognizes human and mouse LCK when phosphorylated on tyrosine 505 (Y505). Y505 is a negative-regulatory site that is phosphorylated by the kinase CSK. LCK is expressed in T cells where it associates with the cytoplasmic tails of the CD4 and CD8 co-receptors on T helper cells and cytotoxic T cells, respectively. When the TCR is engaged, LCK acts to phosphorylate the intracellular chains of CD3 and zeta chains of the TCR complex, allowing the cytoplasmic tyrosine kinase, ZAP-70, to bind to them. LCK then phosphorylates and activates ZAP-70, which then phosphorylates the adaptor protein, LAT, that serves as a docking site for a number of other proteins, including SLP-76, Grb2, and phospholipase C gamma1. Mice lacking expression of LCK show defects in T cell development and function. Applications Reported: This SRRCHA antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This SRRCHA antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of activated human peripheral blood cells. This can be used at 5 µL (0.06 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. 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 (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 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. 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) proteins. 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. Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser. Filtration: 0.2 µm post-manufacturing filtered.
- Reactivity
- Human, Mouse
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- SRRCHA
- Vial size
- 100 Tests
- Concentration
- 5 µL/Test
- Storage
- 4° C, store in dark, DO NOT FREEZE!
Submitted references PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance.
Verma V, Shrimali RK, Ahmad S, Dai W, Wang H, Lu S, Nandre R, Gaur P, Lopez J, Sade-Feldman M, Yizhak K, Bjorgaard SL, Flaherty KT, Wargo JA, Boland GM, Sullivan RJ, Getz G, Hammond SA, Tan M, Qi J, Wong P, Merghoub T, Wolchok J, Hacohen N, Janik JE, Mkrtichyan M, Gupta S, Khleif SN
Nature immunology 2019 Sep;20(9):1231-1243
Nature immunology 2019 Sep;20(9):1231-1243
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Supportive validation
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- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Intracellular staining of untreated (orange histogram) or 5-minute hydrogen peroxide-activated sodium pervanadate-treated (purple histogram) normal human peripheral blood cells with Anti-Human/Mouse phospho-LCK (Y505) PerCP-eFluor® 710 using the Intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Lymphocytes in the CD3+ gate were used for analysis.