Recombinant Human Active Blk Protein, CF
R&D Systems | Catalog # 2679-KS
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Key Product Details
- R&D Systems Sf 9 (baculovirus)-derived Recombinant Human Active Blk Protein (2679-KS)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
Sf 9 (baculovirus)
Accession Number
Applications
Bioactivity
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Product Specifications
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Blk protein
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
N-terminal Sequence Analysis
Using an N terminal GST tag
SDS-PAGE
84 kDa
Activity
The specific activity of Blk was determined to be 110 nmol/min/mg using a poly (Glu:Tyr, 4:1) synthetic peptide substrate (see Activity Assay Protocol).
Scientific Data Images for Recombinant Human Active Blk Protein, CF
Recombinant Human Active Blk Protein SDS-PAGE.
The approximate molecular weight is 84 kDa and the purity is > 90%.Formulation, Preparation, and Storage
2679-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. |
Background: Blk
References
- Dymecki, S.M. et al. (1990) Science 247:332.
- Burkhardt, A.L. et al. (1991) Proc. Natl. Acad. Sci. USA 88:7410.
Long Name
B Lymphoid Tyrosine Kinase
Alternate Names
B lymphocyte kinase, B lymphoid tyrosine kinase, BLK nonreceptor tyrosine kinase, EC 2.7.10.2, MGC10442, MODY11, p55-Blk, tyrosine-protein kinase Blk
Gene Symbol
BLK
UniProt
Additional Blk Products
Product Documents for Recombinant Human Active Blk Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human Active Blk Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Human Active Blk Protein, CF (2679-KS):
Materials
- Active Kinase - Active Blk (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 - 25 mM MOPS, pH 7.2, 12.5 mM beta -glycerolphosphate, 20 mM MgCl2, 25 mM MnCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
- Kinase Dilution Buffer - Kinase Assay Buffer II diluted at a 1:4 ratio (5X dilution) with distilled water.
- 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 (4:1 Glu:Tyr) synthetic peptide substrate diluted in distilled water to a final concentration of 1 mg/mL.
- Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
- Thaw the Active Blk, Kinase Assay Buffer II, Substrate, and Kinase Dilution Buffer on ice.
- In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL:
a. Diluted Active Blk: 10 μL
b. Poly Substrate (1 mg/mL on ice): 5 μL
c. Distilled water: 5μL - 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 water.
- Initiate the reaction with the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL and incubate the mixture in a water bath at 30 °C for 15 minutes.
- After the 15 minute incubation period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
- 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 water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
- Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter
- 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)]