L. Andrew Lee*; Huey Nguyen, Nikki Sitasuwan, and David Miles
Integrated Micro-Chromatography Systems, Irmo, SC
*lee@imcstips.com
Post high throughput discovery, but early-stage mid-throughput sample extraction (>2 mL sample volumes) present a challenge in automation. Many of the liquid handlers are limited to 1 mL or lower pipette volumes and typically designed for high throughput liquid transfers at less than 1 mL. Previous demonstration using 1 mL pipette tips with small resin beds required lengthy binding steps with multiple liquid transfers to process sample volumes > 2 mL, often taking 2-3 hours for 24 samples. Here, we demonstrate an improvement over previous workflows by demonstrating 5 mL pipette tips for protein purifications. We purified poly-histidine tagged GFP using 0.5 mL Ni-IMAC resin bed in 5 mL pipette tip from 24 x 5 mL lysates in less than one hour. The binding kinetics was measured across each aspirating and dispensing step (or binding cycle) across multiple protein concentrations. After identifying binding profiles across different protein concentrations, the elution volumes from 1 to 10x column volume (CV) were adjusted to ensure maximum recovery of 15 mg of GFP bound. Elution volume of 2 mL or 4x CV was selected for testing across multiple protein quantities, comparing between single and double elutions. One elution recovered approximately 80-90% of the bound protein and subsequent second elution added 10-15% to total recovery. This is first demonstration of dispersive solid phase extraction (dSPE) using affinity resin to purify his-tagged protein from 5 mL of lysate. Future work involving multiple pipette head or reagent dispenser for higher throughput will be explored.
