Affinity /ms assay ros Cons get the whole complex doesn t determine direct interactions proteins that purify together are not reliable for small proteins (< 15 kD) likely to share a function affinity tag may interfere with interactions or very sensitive- can detect N 15 with the function of essential proteins copies per cel prone to false positives e.g." sticky"proteins in vivo conditions prone to false negatives can be adapted for wont get every protein every time high-throughput screens complex must survive purification not quantitative
Affinity/ms assay Pros get the whole complex proteins that purify together are likely to share a function very sensitive - can detect ~15 copies per cell in vivo conditions can be adapted for high-throughput screens Cons doesn’t determine direct interactions not reliable for small proteins (< 15 kD) affinity tag may interfere with interactions or with the function of essential proteins prone to false positives, e.g. “sticky” proteins prone to false negatives won’t get every protein every time complex must survive purification not quantitative
Array Detection of Protein-Protein Interactions print onto aldehyde or ①②①②①② Ni surface 3④③④③④ purified peptides or proteins NXN label with fluorescent
Array Detection of Protein-Protein Interactions print onto aldehyde or Ni surface label with fluorescent 1 2 3 4 1 2 3 4 1 2 3 4 purified peptides or proteins N x N dye 1 mm
Highly purified proteins were denatured using GdnHCl and printed onto aldehyde derivatized glass slides using a commercial split pin arrayer. GdnHcl was to prevent homodimerization on the surface 49 human proteins plus 3 duplicates plus 10 yeast proteins were printed in quadruplicate 62 times The 62 proteins were independently labeled with Cy-3 dye and denatured with GdnHCl Peptides were diluted from gdnhcl as they were added to the arrays. Following a brief incubation, slides were washed dried and scanned yielding nxn measurements, in quadruplicate of cc interactions The assay was repeated at concentration ranging from 160 pM to XXX nM
Highly purified proteins were denatured using G dnHCl a nd printed onto aldehydederivatized glass slides using a commercial split pin arrayer. GdnHCl was to prevent homodimerization on the surface. 49 human proteins plus 3 duplicates plus 10 yea st proteins w e r e pri n t ed i n quadr uplicate 62 times. The 62 proteins wer e i ndepende ntly labeled wi t h Cy-3 dye a nd denatur ed with G dnHCl. Peptides were diluted from GdnHCl as they were added to the arrays. Following a brief in c ubation, slide s wer e washed, dri ed a nd sc anned, yielding N x N mea s u r e m ents, in quadruplicate of cc interactions. The assay was repeated at concentration ranging from 160 pM to XXX nM