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HTRF Human Total SHP1 Detection Kit, 10,000 Assay Points

The Total SHP1 kit is designed to monitor the expression level of SHP1, and can be used as a normalization assay for the Phospho-SHP1 (Tyr564) kit.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 64NH1PEG
Unit Size:
Part Number: 64NH1PEH
Unit Size:
  • The Total SHP1 cellular assay monitors the expression level of SHP1, and can be used as a normalization assay with our phospho-SHP1 Y564 kit. Because these phospho and total SHP1 assays are compatible, the two kits can be used in parallel on the same lysates.

    Many cancer cells overexpress checkpoint inhibitor ligands such as PD-L1. PD-L1 binds its counterpart checkpoint inhibitor receptor PD1, present at the surface of T lymphocytes. In turn, the PD1-PDL1 complex recruits and activates inhibitory effectors, such as SHP1 or SHP2. These two phosphatases, which are phosphorylated on Tyr564 and Tyr542 respectively by the kinase Lck, trigger the dephosphorylation of signaling proteins such as ZAP-70 or SLP-76, involved in the T cell activation pathway. Finally, activated SHP1 and SHP2 participate in T cell inactivation.

    Preventing the activation of SHP1 and/or SHP2 by small molecule inhibitors is believed to contribute to restoring the immune response against tumors.

  • Assay Points
    10000
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Infectious Diseases
    Oncology & Inflammation
  • Total-MEK1 assay principle

    The Total-MEK1 assay quantifies the expression level of MEK1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-MEK1 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of MEK1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

    phospho-how-it-works-total-mek1-assay-principle

     

    Total-MEK1 2-plate assay protocol

    The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total MEK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

    phospho-how-it-works-total-mek1-2-plate-assay-protocol

     

    Total-MEK-1-plate assay protocol

    Detection of total MEK with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

    phospho-how-it-works-total-mek-1-plate-assay-protocol

     

  • Validation of the total-MEK1 assay on various cell lines

    Human (Hela, A431, HEK293, Jurkat), murine (NIH 3T3) and Hamster (CHO-K1) cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3 mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging, total MEK1 was detected using 16 µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.

    assay-validation-mek1-total-1

     

    Total-MEK1 assay used to control the phosphorylation status of MEK1

    Hela cells (100,000 cells/well) were activated with EGF for 5 min, using the two-plate assay protocol of the Phospho-MEK1 (Ser218/222) and Total-MEK1 assays. Results obtained show a dose-response increase of MEK1 phosphorylation upon EGF stimulation while MEK1 expression level remains constant.

    assay-validation-mek1-total-2

     

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