Recombinant Human Active ITK (aa 352-620) Protein, CF Summary
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.
|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.|
- Active Kinase - Active ITK (0.1 μg/μL) diluted with Kinase Dilution Buffer IX (1X) and assayed as outlined in sample activity plot. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
- 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 IX (1X) - Kinase Assay Buffer III diluted at a 1:4 ratio (5X dilution) with cold water. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
- ADP-Glo™ Kinase Assay Kit - ATP Solution, 10 mM, ADP Solution, 10 mM, ADP-Glo™ Reagent, and Kinase Detection Reagent.
- Substrate - Myelin basic protein (MBP) diluted in 100 mM MOPS (pH 6.5) buffer to a final concentration of 0.5 mg/mL.
- Thaw the Active ITK, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution at the desired concentration, with Kinase Dilution Buffer IX (1X), in a pre-chilled 96-well plate.
- Prepare a Substrate/ATP mixture as follows (25 μM example):
a. 10 mM ATP Solution: 1 μL
b. Kinase Assay Buffer III (5X): 79 μL
c. Substrate at 0.5 mg/mL: 80 μL
- Transfer the following reaction components prepared in Step 2
to a 384-well opaque plate bringing the reaction volume up to 5
a. 3 μL of diluted Active ITK
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 IX (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)]
The approximate molecular weight is 53 kDa and the purity is > 90%.
ITK is a member of the TEC family of non-receptor tyrosine kinases. ITK is expressed in T cells and is important for T cell development and activation through the antigen receptor. ITK requires prior activation of Lck, ZAP70, and PI3-kinase for efficient activation and shares major substrates with both Lck and ZAP70 (1). ITK knockout mice show multiple effects on T cell development, cytokine production, and T-helper cell differentiation. T cells that lack or express mutant versions of ITK show impaired TCR-induced actin polymerization, cell polarization, and regulation of the signaling events involved in cytoskeletal reorganization (2).
- August, A. et al. (2002) Int. J. Biochem. Cell Biol. 34:1184.
- Finkelstein, L.D. et al. (2004) Trends Cell Biol. 14:443.
No product specific FAQs exist for this product, however you mayView all Proteins and Enzyme FAQs
Reviews for Recombinant Human Active ITK (aa 352-620) Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human Active ITK (aa 352-620) Protein, CF and earn rewards!
Have you used Recombinant Human Active ITK (aa 352-620) 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