Recombinant Human Active Src Protein, CF

Catalog # Availability Size / Price Qty
4595-KS-010
Recombinant Human Active Src Protein, CF SDS-PAGE
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Product Details
Citations (2)
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Recombinant Human Active Src Protein, CF Summary

Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Activity
The activity of Src is 83-113 nmol/min/mg using a poly (Glu:Tyr, 4:1) synthetic peptide substrate (see Activity Assay Protocol).
Source
E. coli-derived human Src protein
Accession # NM_005417
Accession #
N-terminal Sequence
Analysis

Using an N-terminal GST tag

SDS-PAGE
83 kDa

Product Datasheets

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.

4595-KS

Formulation Supplied in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.25 mM DTT, 10 mM glutathione, 0.1 mM EDTA, 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 Src (0.1 μg/μL) diluted with Kinase Dilution Buffer. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer II, pH 7.2 - 25 mM MOPS, 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 prior to use.
  • Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer II diluted 5-fold 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 Src, Kinase Assay Buffer II, 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 Src: 10 μL
    b. Poly Substrate (1 mg/mL; on ice): 5 μL
    c. Distilled or deionized water (on ice): 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 View Larger

The approximate molecular weight is 83 kDa and the average purity is 95%.

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Background: Src

The Src family belongs to non-receptor tyrosine kinases. Src was originally identified as a transforming protein of the Rous Sarcoma Virus (RSV) that had the enzymatic ability to phosphorylate tyrosine in protein substrates (1). Src is over-expressed and activated in a large number of human malignancies and has been linked to the development of cancer and progression to distant metastases (2). In addition to increasing cell proliferation, a key role of Src in cancer seems to be the ability to promote invasion and motility, functions that might contribute to tumor progression.

References
  1. Collett, M.S. et al. (1978) Proc. Natl. Acad. Sci. USA 75:2021.
  2. Jacobs, C. et al. (1983) Cancer Res. 43:1696.
Long Name
v-src Sarcoma [Schmidt-Ruppin A-2] Viral Oncogene Homolog
Entrez Gene IDs
6714 (Human); 20779 (Mouse); 83805 (Rat); 1491925 (Viral)
Alternate Names
ASV; c-Src; EC 2.7.10; EC 2.7.10.2; pp60c-src; Rous sarcoma; RSVgp4; Src; tyrosine kinase pp60c-src; tyrosine-protein kinase SRC-1; v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog; v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)

Citations for Recombinant Human Active Src 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. Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor
    Authors: K Sun, Y Chang, B Zhou, X Wang, L Liu
    Int J Nanomedicine, 2017;12(0):1905-1915.
    Species: N/A
    Sample Types: Protein
    Applications: Bioassay
  2. Sepsis-induced cardiac mitochondrial dysfunction involves altered mitochondrial-localization of tyrosine kinase Src and tyrosine phosphatase SHP2.
    Authors: Zang Q, Martinez B, Yao X, Maass D, Ma L, Wolf S, Minei J
    PLoS ONE, 2012;7(8):e43424.
    Species: Rat
    Sample Types: Mitochondria
    Applications: Bioassay

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