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How things work |
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The recombinant approach
1. The preparation of protein-peptide microarrays for any purpose requires only the amino-acid sequences of the desired protein or peptide. The desired amino-acid sequence is translated into the corresponding DNA sequence and amplified by PCR. The gene of interest is ligated into an appropriate plasmid that contains the Zephyrin gene.
2. The preparation of protein-peptide microarrays for any purpose requires only the amino-acid sequences of the desired protein or peptide. The desired amino-acid sequence is translated into the corresponding DNA sequence and amplified by PCR. The gene of interest is ligated into an appropriate plasmid that contains the Zephyrin gene.
3. The Zephyrin-fused peptide or protein is expressed in E. coli BL21. The cells are lysed, and if necessary the recombinant Zephyrin-fused protein or peptide is affinity-purified on cellulose beads. The expression level is quality-controlled by SDS-PAGE analysis prior to the printing step.
4. The Zephyrin-fused peptide or protein is expressed in E. coli BL21. The cells are lysed, and if necessary the recombinant Zephyrin-fused protein or peptide is affinity-purified on cellulose beads. The expression level is quality-controlled by SDS-PAGE analysis prior to the printing step.
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The chemical-approach – The Topaz™
1. Desired peptides are synthesized chemically. The chemical synthesis includes addition of cysteine to the N- or C-terminus of every peptide.
2. The peptides are dissolved and introduced into 96-well plates. Maleimide-activated Zephyrin is also added into the same 96-well plate. The plate is incubated for 15 min at room temperature. Under approximately neutral conditions the maleimide-activated Zephyrin will form a thioether bond with a sulfhydryl group, i.e., a cysteine residue of a peptide.
3. Nanoliter amounts of desired Zephyrin-conjugated peptides are printed onto a CelluloSlide™ in a highly reproducible, parallel array production process, using customized spotting technology. The slide is immediately ready for assay.
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