Zorbax c8 2.5 50 mm column

Quantification of unreacted soluble protein in the aggregation mixture was followed by periodically removing aliquots (~80 μL) from ongoing aggregation reactions followed by centrifugation at 160,000×g and injecting the supernatant (approx. 20 μL) in duplicates into an analytical reverse phase column (Agilent Zorbax-C8 2.5 × 50 mm column). Protein samples were run against a gradient of acetonitrile (0–100%) in 0.1% TFA maintained at 300 μL·min-1 flow rate in an Agilent HPLC system (1100 series). The wavelength of the UV detector was fixed at 280 nm. HPLC grade solvents (Water and Acetonitrile) used as mobile phase in the reverse phase HPLC system were obtained from Fisher Chemicals. Concentration of the unreacted soluble protein was determined from integrated A280 nm peaks using standard curves individually acquired for each protein.³⁷

Quantification of unreacted soluble protein in the aggregation mixture was followed by periodically removing aliquots (~80 μL) from ongoing aggregation reactions followed by centrifugation at 160,000×g for 45 minutes and injecting two technical replicates of the supernatant (approx. 20 μL) into an analytical reverse phase column (Agilent Zorbax-C8 2.5 × 50 mm column). Protein samples were run against a gradient of Acetonitrile (0–100%) in 0.1% TFA maintained at 300 μL·min⁻¹ flow rate in a Shimadzu 20 HPLC system. The wavelength of the UV detector was fixed at 280 nm. HPLC grade solvents (Water and Acetonitrile) used as mobile phase in the reverse phase HPLC system were obtained from Fisher Chemicals. Concentration of the unreacted soluble protein was determined from integrated peaks at absorbance 280 nm using standard curves individually acquired for each protein as described previously [21 (link), 22 (link)].