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K-LISA™ Assay Kits for Akt, Checkpoint kinases, and Protein Tyrosine Kinases  | | | | | | | | What is K-LISA? K-LISA stands for Kinase Activity ELISA. It is a method to measure kinase activity semi-quantitatively using enzyme-linked immunosorbent assays (ELISAs) with phospho-specific antibodies. The kinase can be purified, semi-purified, or present in a cell extract, and it is incubated with ATP and the provided peptide or polypeptide substrate. Inhibitors or activators can be added to study their effects on kinase activity. The kits combine the sensitivity and ease of use of ELISA kits with the versatility of an activity assay. K-LISA kits are 96-well format and can be used for: | | | | | | • Inhibitor screening
• Monitoring activation effects
• Mutational effects
• In vitro functional studies
• Monitoring cellular signaling events |
| | |  Diagram: How K-LISA works: | K-LISA procedure
The sample containing the kinase to be assayed is incubated with ATP and the polypeptide or peptide substrate. Inhibitors or activators (both available separately) can be added for screening or functional studies. An HRP-antibody conjugate is added (in some kits a detection antibody is also required), and this is followed by addition of the TMB substrate and detection at 450 nm. Each K-LISA kit is optimized to assay kinase activity rapidly and sensitively. | | |  | K-LISA Products Available:
| Product Name | Kinase Assay Specificity* | Range | Cat. No. | Related Products | | K-LISA Akt Activity Kit | Akt | 6-100 mU | | | | K-LISA PTK Screening Kit | Tyrosine Kinases | Varies† | | | | K-LISA Checkpoint Activity Kit | Chk1
Chk2 | 25-725 µU
260 µU – 26 mU | | |
| | | | NEW K-LISA PTK Screening Kit Single Substrate Plates: | | Cat. No. | Product | Suitable for Screening | Suggested Inhibitors | Inhibitor Cat. No. | | 539702 | K-LISA PTK E4Y Reaction Plate | EGFR | BP1Q-II, 8 pM | | | PD153035, 25 pM | 234490, 234491 | | b-InsR | AG 538, 60 nM | 658403 | | AGL 2263, 400 nM | 121850 | | Lck | PP2, 4 nM | 529573, 529576 | | Src Kinase Inhibitor I, 88 nM | 567805 | | ZAP70 | Piceatannol, 10 uM | 527948 | | PP1, 100 uM | | | 539703 | K-LISA PTK EAY Reaction Plate | FLT3 | GTP-14564, 300 nM | 371806 | | Abl | Imatinib, 33 nM | | | Lck | PP2, 4 nM | 529573 | | Src Kinase Inhibitor I, 88 nM | 567805 | | 539704 | K-LISA PTK EY Reaction Plate | Src | Src Kinase Inhibitor I, 44 nM | 567805 | | SU6656, 280 nM | 572635, 572636 | | Lavendustin A, 11 nM | 428150 | | Lavendustin C, 200 nM | 234450 | | Lck | PP2, 4 nM | 529573 | | Src Kinase Inhibitor I, 88 nM | 567805 |
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| Data for several applications is shown below: | | Figure 1. Typical assay results using cell lysates (K-LISA PTK Screening Kit, Cat. No. 539701): | | |  | A431, HeLa and HEK293 cells were grown at 37 °C, 5% CO2 in DMEM supplemented with 10% FBS to 90% confluency in T225 flasks. Cell lysates were prepared using 2-3 mL of PhosphoSafe™ Extraction Reagent (Cat. No. 71296) and stored at –70 °C until use. A431 lysates significantly phosphorylate E4Y and EY substrates, HeLa lysates favor the EAY and EY substrates, while HEK293 lysates predominantly phosphorylate the EAY substrate. Values shown have been corrected for background and are the averages of triplicate measurements. Backgrounds are presented as black columns. |
| | | | Figure 2: Activity of Akt immunoprecipitated from cell lysate (K-LISA AKT Activity Kit, Cat. No. CB0A19): | | | | Near-confluent MCF-7 cells were stimulated with IGF-1 (100 ng/ml) or IGF-1 (100 ng/ml) and 17b-estradiol (500 nM) for 30 min at 37°C in a CO2 incubator. For inhibition of Akt, cells were first pre-incubated at 37°C in a CO2 incubator for 15 min in the presence of cell-permeable Akt Inhibitor II followed by stimulation with IGF-1 (100 ng/ml) for 30 min at 37°C. Cell lysates were prepared using 0.5 ml PhosphoSafe™ Extraction Buffer per 10 cm cell culture dish, per the standard protocol. Cell lysates were either used immediately or stored at –70°C until use. Equal amounts of total protein (1.5 mg) were immuno-precipitated and activity was determined. |
| | Figure 3: Titration of Chk1, His•Tag®, Human, Recombinant activity (K-LISA Checkpoint Activity Kit, Cat. No. CBA020): | | | | | | | | | Figure 4: Titration of Staurosporine Inhibition of 2 ng of Chk1, His•Tag, Human, Recombinant (K-LISA Checkpoint Activity Kit, Cat. No. CBA020): | | | | | More Related Products: | Kinase Inhibitors
ProteoEnrich™ ATP Binders
PhosphoSafe™ Extraction Buffer | | | |
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