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Abstract
This paper presents rapid protein purification and proteolysis methods that integrate membrane technology and pipet tips. Pushing a protein-containing solution through a protease-modified membrane at the end of a pipet tip digests proteins in 30 s or less, and the short proteolysis time avoids reformation of disulfide bonds to enable tryptic digestion without alkylation of cysteine residues. Moreover, proteolysis is more complete than digestion for 30 min in solution. Antibody digestion at the end of a pipet tip leads to 100% peptide coverage in MS analyses. Similarly, when membranes contain Ni(2+) complexes, pipetting aqueous polyhistidine-tagged protein through the membrane and subsequent rinsing and elution yield purified polyhistidine-tagged protein in 2 min. These applications demonstrate the potential for combining functional membranes and pipet tips for rapid sample purification and pretreatment.
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