Quantitative Measurement of Anti c-myc Epitope Peptide Binding
Chip Design
- Epitope (known) EQKLISEEDL
- HA, c-myc, VSVG, and flag epitope and other peptide sequences (see figure 3 below for detail)
- >3 redundancy
- CV < 30%
Assay
- Antibody: anti-c-myc (1 ng/ml)
- 200 µl TBS
- Temp: 4oC
- Reaction time: 1 hour
Detection
Results
- Figure 1 - Fluorescent images of 4K peptide chip for 3 different experiments
Figure 1
- On chips of more complex sequence design, IgG shows cross interactions with certain peptide sequences, but this potentially can be reduced by a better blocking procedure (experiment #3 of
improved block condition compared to experiment #1)
- The peptide chips can be reused multiple times. Experiment #3 was followed by anti VSVG binding assay.
- Anti c-myc, unlike anti HA, AFM2, and VSVG, exhibits higher
cross reactivity and the epitope sequence has too low binding affinity to be detected.
Figure 2
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EQKLISEEDL
Red: residues can be replaced by an Ala and give higher binding affinity for the 10-mer epitope.
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EQKLISEEDL
Blue: residues dispensable from
both ends and gives higher binding affinity.
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- Note that anti c-myc did not show binding to the epitope sequence and many of its sequence variants.
- The figure only displays the few positive binders.
- Anti c-myc has higher cross-reactivity compared to anti HA, AFM2, and anti VSVG
- Figure 3 - Chip sequence information detail
Figure 3
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