Two-component atomic force microscopy recognition imaging of complex samples.
Biological complexes are typically multisubunit in nature and the processes in which they participate often involve protein compositional changes in themselves and/or their target substrates. Being able to identify more than one type of protein in complex samples and to track compositional changes during processes would thus be very useful. Toward this goal, we describe here a single-molecule technique that can simultaneously identify two types of proteins in compositionally complex samples. It is an adaptation of the recently developed atomic force microscopy (AFM) recognition imaging technique but involves the tethering of two different types of antibodies to the AFM tip and sequential blocking with appropriate antigenic peptides to distinguish the recognition from each antibody. The approach is shown to be capable of simultaneously identifying in a single AFM image two specific components, BRG1 and beta-actin, of the human Swi-Snf ATP-dependent nucleosome remodeling complex and two types of histones, H2A and H3, in chromatin samples.
Wang
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Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, USA.
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D000199:Actins / Q000032:analysis; D002843:Chromatin / Q000737:chemistry; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000032:analysis; D006657:Histones / Q000032:analysis; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry*; D009687:Nuclear Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry
D000199:Actins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011506:Proteins; C445179:SWI-SNF-B chromatin-remodeling complex; D014157:Transcription Factors; D004247:DNA; C084108:SMARCA4 protein, human; D004265:DNA Helicases