PO0018 Expanding Functionality of Automated Liquid Handling Systems by Incorporating INtip Chemistriesfor High Throughput Protein Purification
来源:IMCS
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作者:P. Nikki Sitasuwan, Todd Mullis, Caleb Schlacter, Sunil Hwang, L. Andrew Lee*
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发布时间: 2168天前
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3364 次浏览
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Protein purification is a multi-step process that incorporates various resins and consumables to achieve the desired outcome.For instance, recombinant protein purification is typically achieved using an affinity resin such as Ni-NTA followed by bufferexchange to remove the excess salts. Other affinity-based purification methods are also similar in that the IgG purified with Protein A or G utilize basic buffer to neutralize the acidic elution, but the final storage buffer may require alternativeformulations. Here, we integrated Hamilton Microlab STAR automated liquid handling system with IMCStips to achieve highthroughput protein purification followed by multiple workflow steps to alleviate switching equipment or consumables foreach step. The first workflow is the purification of the recombinant protein by Ni-NTA, followed by buffer exchange on SizeX150 to remove the excess imidazole salts. The purified protein is then aliquoted for protein quantification. The workflowdemonstrates turn-key approach from cell lysate to quantified, purified proteins within less than 2 hours per batch. Thesecond workflow is the characterization of IgG afer Protein A purification followed by denaturation, reduction, alkylation,buffer exchange on SizeX followed by trypsin digestion for multi-attribute monitoring. The third is the reverse phase peptidedesalting and phosphopeptide enrichment on a single workflow. And the last workflow is the use of streptavidin beads inside the pipette tip to capture biotinylated anti-Fc antibody, followed by pull-down of Avastin in mouse serum. The elutedsample is then reduced, alkylated, trypsin digested for quantifying biotherapeutic antibody in mouse sera. These integratedworkflows with IMCStips demonstrate purification of different proteins using different INtip™ chemistries and the flexibility ofthe Hamilton Microlab STAR to achieve a variety of processes on a single platform to achieve high throughput workflows withminimal human intervention.
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