Recombinant Human Active Src Protein, CF
Recombinant Human Active Src Protein, CF Summary
Product Specifications
Analysis
Using an N-terminal GST tag
Complete Your Research
Product Datasheets
Carrier Free
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.
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, 10 mM Glutathione, 0.1 mM EDTA, 0.25 mM DTT, 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
- Active Kinase - Active Src (0.1 μg/μL) diluted with Kinase Dilution Buffer X (1X). Note: These are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active Src 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 (1X) - 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 2.5 mM MnCl2, and 0.1 mg/mL BSA. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
- ADP-Glo™ Kinase Assay Kit - 10 mM ATP Solution, 10 mM ADP Solution, ADP-Glo™ Reagent, and Kinase Detection Reagent.
- Substrate - Src synthetic peptide substrate (KVEKIGEGTYGVVYK) diluted in 20 mM Tris-HCl, pH 7.5 to a final concentration of 1 mg/mL.
- Cofactor: 2.5 M MnCl2 - Diluted to a working concentration of 1 M in distilled water.
- Thaw the Active Src, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution with Kinase Dilution Buffer X (1X), in a pre-chilled 96-well plate.
- 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 - Transfer the following reaction components prepared in Step 2
to a 384-well opaque plate, bringing the reaction volume up to 5
μL:
a. 3 μL of diluted Active Src
b. 2 μL of Substrate/ATP mix as prepared in Step 2. This initiates the reaction. - 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 (1X).
- Incubate at ambient temperature for 40 minutes.
- 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.
- 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.
- Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
- 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/min/μg or nmol/min/mg)
Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in min) x (Enzyme amount in μg or mg)]
Scientific Data
Reconstitution Calculator
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.
- Collett, M.S. et al. (1978) Proc. Natl. Acad. Sci. USA 75:2021.
- Jacobs, C. et al. (1983) Cancer Res. 43:1696.
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.
3
Citations: Showing 1 - 3
Filter your results:
Filter by:
-
Cell adhesion suppresses autophagy via Src/FAK-mediated phosphorylation and inhibition of AMPK
Authors: M Zhao, D Finlay, E Kwong, R Liddington, B Viollet, N Sasaoka, K Vuori
Cellular Signalling, 2021;89(0):110170.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
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 -
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
FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Human Active Src Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human Active Src Protein, CF and earn rewards!
Have you used Recombinant Human Active Src Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥1250 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
