title: Rapid detection and quantification of specific proteins by immunodepletion and microfluidic separation.
abstract: Conventional immunoblotting techniques are labor intensive, time consuming and rely on the elution of target protein after depletion. Here we describe a new method for detection and quantification of proteins, independent of washing and elution. In this method, the target protein is first captured by immunodepletion with antibody-coated microbeads. In the second step, both the supernatant after immunodepletion and the untreated protein sample are directly analyzed by microfluidic electrophoresis without further processing. Subsequently, the detection and quantification are performed by comparing the electropherograms of these two samples. This method was tested using an Escherichia coli lysate with a FLAG-tagged protein and anti-FLAG magnetic beads. An incubation of as short as one min was sufficient for detectable depletion (66%) by microchip electrophoresis. Longer incubation (up to 60 min) resulted in more depletion of the target band (82%). Our results show that only 19% of the target is recovered after elution from the beads. By eliminating multiple wash and elution steps, our method is faster, less labor intensive, and highly reproducible. The target protein can still be easily identified even in the case of nonspecific binding at low concentrations. This work highlights the advantages of integrating immunodepletion techniques on a microfluidic platform.
authors.affiliation: Center for Biomedical Engineering, School of Engineering and Division of Biology & Medicine, Brown University, Providence, RI 02912, USA.
authors.forename: Glareh
authors.initials: G
authors.lastname: Azadi
authors.forename: Eric
authors.initials: E
authors.lastname: Gustafson
authors.forename: Gary M
authors.initials: GM
authors.lastname: Wessel
authors.forename: Anubhav
authors.initials: A
authors.lastname: Tripathi
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D046209:Electrophoresis, Microchip / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins