A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.
Extract and amplify human kinase gene sequences via PCR to generate template DNA for protein array printing.
Apply aminosilane coating to glass slides and prepare purified DNA samples for microarray spotting.
Spot DNA templates onto coated slides using a microarrayer and verify DNA loading via fluorescence.
Perform cell-free transcription–translation directly on printed DNA to generate arrayed human proteins.
Use fluorescent antibodies to confirm successful protein expression at each array spot.
Incubate arrayed proteins with tyrosine kinase inhibitors and detect inhibitor binding via immunofluorescence.
Quantify and visualize expression levels across the array to assess NAPPA platform performance.
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