Example: Screen of the AD-array with rPc19 as a bait This is the activation domain array consisting of 6144 yeast colonies each expressing a different fusion of the gal4 activation domain and one of the yeast ORFs The array is mated to the rPC19 bait and diploids are pinned to selective plates This is a set of selective plates (-- His-Ade)on which only colonies grow that contain RPC19-interacting proteins Stan Field s web site http://depts.washingtonedu/sfields/images/rpc19.html Courtesy of stanley Fields. Used with permission
Stan Field’s web site http://depts.washington.edu/sfields/images/RPC19.html Courtesy of Stanley Fields. Used with permission
Additional cons"when you do a large scale 2-hybrid screen CR amplification gives mutations -generally dont sequence everything to confirm Cloning transformation inefficiencies If baits are pooled slow-growing cells will lose to faster ones, giving false negatives All VS all assay contains many implausible interactions - proteins that arent Co-localized or expressed at the same time Can only sequence a small fraction of the positive clones High-throughput Y2H screens miss as many as 90% of Y2H interactions observed in focused, small-scale studies
Additional “cons” when you do a large scale 2-hybrid screen PCR amplification gives mutations - generally don’t sequence everything to confirm! Cloning & transformation inefficiencies If baits are pooled, slow-growing cells will lose to faster ones, giving false negatives. All vs. all assay contains many implausible interactions - proteins that aren’t co-localized or expressed at the same time. Can only sequence a small fraction of the positive clones. High-throughput Y2H screens miss as many as 90% of Y2H interactions observed in focused, small-scale studies!
Affinity Purification What do you mean by an " interaction? Most proteins interact with several other proteins(estimate 2-10) Many proteins in the cell are found in complexes. for some purposes knowing the identities of the members of the clusters is as useful, or more useful, than knowing the directly interacting partners. Affinity purification is a method for characterizing the clusters directly, rather than one interaction at a time
Affinity Purification What do you mean by an “interaction”? Most proteins interact with several other proteins (estimate 2-10). Many proteins in the cell are found in complexes. For some purposes, knowing the identities of the members of the clusters is as useful, or more useful, than knowing the directly interacting partners. Affinity purification is a method for characterizing the clusters directly, rather than one interaction at a time
Affinity Purification/Mass spectrometry DNA encodes bait tag a C BAIT bait expressed in cell forms part of a se cell, fish for complex complex with affinity column that binds a separate affinity tac a, b, d, e bait C BAIT by sds page gel extract bands identities digest with trypsin proteins PEPTIDES in the mass spec t complex database search
Affinity Purification/Mass spectrometry affinity tag BAIT a b c d e BAIT a b c d e DNA encodes bait + tag bait expressed in cell forms part of a complex complex with affinity the tag separate a,b,c,d,e,BAIT extract bands, digest with trypsin PEPTIDES mass spec + identities of proteins in the complex lyse cell, fish for column that binds by SDS PAGE gel database search
Affinity purification /mass spectrometry for an entire genome Gavin et al. Nature(2002)415, 141-147; Cellzome 1, 167 bait proteins TAP tag inserted at 3 end of gene proteins under endogenous promoter 2 rounds of purification 232 distinct complexes with 2 to 83 proteins per complex new cellular role proposed for 344 proteins o assess confidence Repeat the experiment-only 70% reproducible using the same bait Use different proteins in the complex as the bait, see if you recover the same proteins in the complex. Ho et al. Nature(2002)415, 180-183 MDS Proteomics 725 bait proteins; 1, 578 interacting proteins FLAG tag proteins transiently overe expressed To assess confidence 74% of interactions reproducible in small scale co-IP/blot
Affinity purification/mass spectrometry for an entire genome Gavin et al. Nature (2002) 415, 141-147; Cellzome 1,167 bait proteins TAP tag inserted at 3’ end of gene; proteins under endogenous promoter 2 rounds of purification 232 distinct complexes with 2 to 83 proteins per complex new cellular role proposed for 344 proteins To assess confidence: Repeat the experiment - only 70% reproducible using the same bait Use different proteins in the complex as the bait, see if you recover the same proteins in the complex. Ho et al. Nature (2002) 415, 180-183; MDS Proteomics 725 bait proteins; 1,578 interacting proteins FLAG tag, proteins transiently overexpressed To assess confidence: 74% of interactions reproducible in small scale co-IP/blot