Recombinant Human Active ZAP70 Protein, CF

Catalog # Availability Size / Price Qty
3709-KS-010
Recombinant Human Active ZAP70 Protein SDS-PAGE
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Citations (2)
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Recombinant Human Active ZAP70 Protein, CF Summary

Product Specifications

Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Activity
The specific activity of ZAP70 was determined to be 13 nmol/min/mg using a Poly (Glu:Tyr, 4:1) synthetic peptide substrate.
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human ZAP70 protein
Accession #
N-terminal Sequence
Analysis
Using an N-terminal GST tag
SDS-PAGE
96 kDa

Product Datasheets

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3709-KS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

3709-KS

Formulation Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: This product is stable at ≤ ‑70 °C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.

Assay Procedure

Materials
  • Active Kinase - Active ZAP70 (0.1 μg/μL) diluted with Kinase Dilution Buffer X and assayed as outlined in sample activity plot. Note: These are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active ZAP70 for optimal results.
  • Kinase Assay Buffer III (5X) - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
  • Kinase Dilution Buffer X - 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 2.5 mM MnCl2, and 0.1 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 50 μM.
  • ADP-Glo™ Kinase Assay Kit - 10 mM of ATP Solution, 10 mM of ADP Solution, ADP-Glo™ Reagent, and Kinase Detection Reagent.
  • Substrate - Poly (4:1 Glu:Tyr) synthetic peptide substrate diluted in 25 mM Tris-HCl to a final concentration of 1 mg/mL.
  • Cofactor: 2.5 M MnCl2 - Diluted in distilled water to a working concentration of 1 M.
  1. Thaw the Active ZAP70, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution with Kinase Dilution Buffer X at the desired concentration, in a pre-chilled 96-well plate.
  2. Prepare a Substrate/ATP mixture as follows (25 μM ATP example):
    a. 10 mM ATP Solution: 1 μL
    b. Kinase Assay Buffer III (5X): 78 μL
    c. Substrate at 1 mg/mL: 80 μL
    d. 1 M MnCl2: 1 μL
  3. Transfer the following reaction components prepared in Steps 1 and 2 to a 384-well opaque plate, bringing the reaction volume up to 5 μL:
    a. Diluted Active ZAP70: 3 μL
    b. Substrate/ATP (mix as prepared in Step 2). This initiates the reaction: 2 μL
  4. Set up the blank control as outlined in Step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer X.
  5. Incubate at ambient temperature for 40 minutes.
  6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo™ Reagent. Spin down and shake the 384-well plate. Then incubate the reaction mixture for another 40 minutes at ambient temperature.
  7. Then add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature.
  8. Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
  9. Determine the corrected activity (RLU) by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below.


    Calculation of Specific Activity of ADP (RLU/pmol)
    From ADP standard curve, determine RLU/pmol of ADP

    Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)]

Scientific Data

SDS-PAGE Recombinant Human Active ZAP70 Protein SDS-PAGE View Larger

The approximate molecular weight is 96 kDa and the purity is > 90%.

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Background: ZAP70

ZAP70 is a non-receptor protein tyrosine kinase (part of the Syk/ZAP70 family) that is involved in signaling by the T-cell antigen receptor (TCR). Ligation of the TCR/CD3 receptor in Jurkat T-cells induces phosphoprotein complexes that contain ZAP70 (1). TCR zeta chains are initially phosphorylated by p56Lck that lead to the recruitment of ZAP70 via its SH2 domain. ZAP70 in turn phosphorylates other proteins in the TCR-phosphoprotein complex. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex (2).

References
  1. Duplay, P. et al. (1994) J. Exp. Med. 179:1163.
  2. Chan, A.C. et al. (1994) J. Immunol. 152:4758.
Long Name
Zeta-chain [TCR] Associated Protein Kinase 70 kDa
Entrez Gene IDs
7535 (Human); 22637 (Mouse); 301348 (Rat)
Alternate Names
70 kDa zeta-associated protein; EC 2.7.10; EC 2.7.10.2; mrtle; mur; SRK; SRKFLJ17679; STD; STDFLJ17670; Syk-related tyrosine kinase; tyrosine-protein kinase ZAP-70; TZK; ZAP70; ZAP-70; zeta-chain (TCR) associated protein kinase (70 kD); zeta-chain (TCR) associated protein kinase 70kDa; zeta-chain associated protein kinase, 70kD

Citations for Recombinant Human Active ZAP70 Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

2 Citations: Showing 1 - 2
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  1. Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70and destabilization of the signalosome
    Authors: ML Giardino T, D Dutta, PR Mittelstad, J Guha, MM Gaida, K Fish, VA Barr, IO Akpan, LE Samelson, HD Tagad, S Debnath, LM Miller Jen, E Appella, JD Ashwell
    Proc. Natl. Acad. Sci. U.S.A., 2018;0(0):.
  2. Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation
    Authors: MS Alam, MM Gaida, S Debnath, HD Tagad, LM Miller Jen, E Appella, MJ Rahman, JD Ashwell
    PLoS Biol., 2018;16(1):e2004111.
    Species: Mouse
    Sample Types: Recombinant Protein
    Applications: Bioassay

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