TY - JOUR
T1 - Kinase inhibitor screening in self-assembled human protein microarrays
AU - Festa, Fernanda
AU - Labaer, Joshua
N1 - Funding Information:
The authors would like to thank everyone at the LaBaer’s lab for their help and criticism during the development of the project. This project was supported by the NIH grant U01CA117374, U01AI077883 and Virginia G. Piper Foundation.
Publisher Copyright:
© 2019 Journal of Visualized Experiments.
PY - 2019/10
Y1 - 2019/10
N2 - The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.
AB - The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.
KW - Biochemistry
KW - Drug screening
KW - High-throughput screening
KW - Issue 152
KW - NAPPA protein microarray
KW - Protein kinase assay
KW - Screen of kinase inhibitors
KW - Self-assembled protein microarrays
KW - Tyrosine kinase inhibitors
UR - http://www.scopus.com/inward/record.url?scp=85074742476&partnerID=8YFLogxK
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U2 - 10.3791/59886
DO - 10.3791/59886
M3 - Article
C2 - 31710025
AN - SCOPUS:85074742476
VL - 2019
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
SN - 1940-087X
IS - 152
M1 - e59886
ER -