Recombinant Human Active SYK Protein, CF

Catalog # Availability Size / Price Qty
4594-KS-010

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Recombinant Human Active SYK Protein SDS-PAGE
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Recombinant Human Active SYK Protein, CF Summary

Product Specifications

Purity
>85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Activity
The specific activity of SYK is typically 79-107 nmol/min/mg using a poly (Glu:Tyr, 4:1) synthetic peptide substrate.
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human SYK protein
Accession #
N-terminal Sequence
Analysis

Using an N-terminal GST tag

SDS-PAGE
100 kDa

Product Datasheets

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4594-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.

4594-KS

Formulation Supplied in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.25 mM DTT, 0.1 mM EDTA, 10 mM Glutathione, 0.1 mM PMSF, and 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 SYK (0.1 μg/μL) diluted with Kinase Dilution Buffer IV. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer II - 25 mM MOPS, pH 7.2, 12.5 mM beta -glycerolphosphate, 20 mM MgCl2, 12.5 mM MnCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer II prior to use.
  • Kinase Dilution Buffer IV - Kinase Assay Buffer II diluted at a 1:4 ratio (5X dilution) with a 50 ng/μL BSA solution.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer II. Store 200 μL aliquots at ≤ -20 °C.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer II. Store 1 mL aliquots at ≤ -20 °C.
  • Substrate - Poly (Glu:Tyr; 4:1) synthetic peptide diluted in distilled or deionized water to a final concentration of 1 mg/mL.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active SYK, Kinase Assay Buffer, Substrate, and Kinase Dilution Buffer on ice.
  3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL:
    a. Diluted Active SYK: 10 μL
    b. Stock solution of Substrate (1 mg/mL): 5 μL
    c. Distilled or deionized water (2-4 °C): 5 μL
  4. Set up the blank control as outlined in Step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
  5. Initiate the reaction with the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  6. After the 15 minute incubation, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
  7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. Determine the corrected cpm by subtracting the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below:


    Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmol of ATP (in 5 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)

    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]

Data Image

SDS-PAGE Recombinant Human Active SYK Protein SDS-PAGE. View Larger

The approximate molecular weight is 100 kDa and the purity is > 85%.

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Background: SYK

SYK is a non-receptor protein tyrosine kinase that is widely expressed in hematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis. In B cells, SYK plays a crucial role in intracellular signal transduction induced by oxidative stress as well as antigen receptor engagement (1). SYK has been shown to act as a potential tumor suppressor in breast cancer. Absence of SYK protein in primary breast tumors is correlated with poor outcomes. SYK deficient cells have increased motility that is restored to normalcy by replacement with wild-type SYK (2).

References
  1. Takano, T. et al. (2002) Antioxid. Redox. Signal. 4:533.
  2. Navara, C.S. et al. (2004) Curr. Pharm. Des. 10:1739.
Long Name
Spleen Tyrosine Kinase
Entrez Gene IDs
6850 (Human); 20963 (Mouse); 25155 (Rat)
Alternate Names
DKFZp313N1010; EC 2.7.10; EC 2.7.10.2; FLJ25043; spleen tyrosine kinaseFLJ37489; SYK; tyrosine-protein kinase SYK

Citation for Recombinant Human Active SYK 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.

1 Citation: Showing 1 - 1

  1. STING-Mediated Interferon Induction by Herpes Simplex Virus 1 Requires the Protein Tyrosine Kinase Syk
    Authors: C Wang, N Sharma, M Veleeparam, PM Kessler, B Willard, GC Sen
    MBio, 2021;12(6):e0322821.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay

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