| Protein kinases are enzymes that phosphorylate target proteins, typically resulting in target activation. Because phosphorylation events regulate myriad signal transduction/disease pathways, kinases are attractive drug targets and the subject of intense research. Over the past 15 years, highly sensitive and specific methods have been created to monitor the level and activity of several kinases. As a result, a large selection of kinase assay and detection kits is available, offering increased flexibility for researching signaling pathways and kinase function. Due to the wide variety of kits, it is important to understand the format, strengths, limitations, and requirements of each assay. Enzyme-linked immunosorbent assays (ELISAs) can be used to quantitate kinase levels and evaluate their activities. ELISAs are popular because they are generally fast, inexpensive, easily accommodate multiple samples, and do not require highly specialized equipment. By incorporating pan (recognize protein regardless of phosphorylation state) and phospho-specific antibodies, ELISA-based assays can be used to measure total protein and activated protein levels. ELISA kits offer an indirect measurement of kinase activity, but because they are antibody-based, they are specific, sensitive, and can be used with a variety of sample types. K-LISA™ kits utilize a peptide or polypeptide substrate and phospho-specific antibodies to directly measure kinase activity in a wide variety of samples, including cell lysates and purified or partially purified kinase samples. K-LISA kits combine the ease of use and sensitivity of ELISA kits with direct measurement of kinase activity and can accommodate inhibitor, effector, and immunoprecipitation (IP) based studies (Figures 1 & 2). Activity assays utilizing colorimetric or Western blot detection are also available. These kits vary in the sample type, applications, and equipment needed. Refer to the Table on the Activity Assays section and read the protocol before choosing.
Having a wide variety of kinase assay and detection kits is important because different stages of kinase research have different requirements. In the early discovery phases of kinase research, the specificity that an antibody-based technique such as ELISA provides is needed. In the later analysis stages, higher speed is required, and peptide-based methods are more suitable. The methods can be complementary to each other in a protein kinase research project, and can also utilize the same companion products such as inhibitors, substrates, and enzymes. Use the Protein Kinase Assay & Detection Kits brochure and this web resource to determine which kits and products will meet your needs.
How to choose a protein kinase kit
When choosing a kinase kit, first consider your goal and determine which measurements would serve the purpose. For example, if you would like to measure active Akt levels, you could measure the amount of phosphorylated Akt using a PhosphoDetect™ ELISA kit or you could measure Akt activity directly using a K-LISA™ Activity Kit. If a kit is not available for your protein kinase of interest, consider measuring the activity indirectly by measuring the phosphorylation of one of its targets using a PhosphoDetect ELISA kit or antibody.
Next, consider the sample available for testing. Cell extracts contain a plethora of kinases, so using an immunoprecipitating (IP) antibody approach or a sandwich-type PhosphoDetect ELISA kit would be most preferable since activity kits may contain a nonexclusive substrate. Functional studies with purified enzymes are obviously best suited for activity assays such as K-LISA or TruLight because they directly measure kinase activity and allow for attenuation by an inhibitor or stimulation by an activator. If you need to screen many kinases or inhibitors, choose a TruLight Kinase Assay, because they do not require mixing steps, making them easier to use and fully automatable. An additional consideration is the mechanism of the inhibitor/activator utilized. For example, most specific Akt inhibitors do not directly affect the activity of an enzyme, but affect its regulation, so they would not be suitable for use with a kit that directly measures Akt activity. A more general, direct-acting inhibitor such as Staurosporine (Cat. No. 569397) should be used with a kit such as the K-LISA Akt Activity Kit. Check the sensitivity and range of the assay to ensure that it matches the expected level of kinase in your sample. Finally, make sure that the species reactivity is applicable for your sample. In general, species reactivity will be broader for activity assays than for ELISA kits. However, any step involving antibody detection (e.g., immunoprecipitation) may limit the species reactivity of an activity assay to the species reactivity of the antibody. Refer to the kinase kit selection guide table on the bottom of this page for general guidelines and additional information as well as to the Technical Tips section. |
