Multi-Step Protein Purification

 

Downstream Size Exclusion/Gel Filtration
Starting material will be partially purified protein concentrated in a suitable volume of load buffer. The protein sample will be injected on to an equilibrated gel filtration column (Superdex 75 or 200; Sephacryl 300) and the column will be developed with equilibration buffer and fractions collected. Fractions will be analyzed for total protein (Bradford) and purity by SDS-PAGE. Results (SDS-gel images) will be communicated with customer for decisions on pooling of best fractions. Pooling and concentration (Amicon Ultra) of one peak is included with service.

Delivered: Purified protein concentrated in column buffer. Experimental summary including SDS-gel images
Time: 2-4 days.

 

Downstream Ion Exchange Chromatography (DEAE / Q / SP / CM)
Starting material will be partially purified protein in a suitable low ionic strength buffer to bind IEX-Sepharose. Protein will be passed over 5-10 mL of resin (capacity ~1g protein) and washed with load buffer to baseline (OD280nm). Bound protein will be eluted by development of column with a linear sodium chloride gradient. Fractions will be analyzed for total protein (Bradford) and purity by SDS-PAGE. Elution profile results (SDS-gel) will be communicated to customer for pooling of fractions. Buffer exchange and concentration for additional charge (see below).

Delivered: Purified protein in elution buffer with final amount determined by efficiency of binding and elution to from IEX-Sepharose.
Time: 4-6 days.

 

Downstream Hydrophobic Interaction Chromatography (Octyl or Phenyl Sepharose)
Use of an HIC purification step requires data indicating that the target protein is (i) soluble in 1-2 M ammonium sulfate, (ii) binds to HIC resin, and (iii) recoverable from the HIC resin. A scouting experiment is recommended before use of this chromatographic method. The ionic strength of a partially purified protein solution (from a previous purification step) will adjusted by addition of ammonium sulfate to ~200mg/mL (~33% saturation) and allowed to equilibrate for 1h with gentle mixing. Any precipitated protein will be removed by centrifugation (20000g; 1h) and the clarified protein solution mixed with 25ml of HIC resin for 2h. The capacity of 25ml phenyl or octyl-Sepharose is approximately 750mg of protein. The protein-bound HIC resin will be transferred to an FPLC column and washed with high ionic strength buffer to baseline (OD280nm). The column will be developed by applying a negative gradient of ammonium sulfate over 3-5 column volumes. Fractions will be analyzed for total protein (Bradford) and purity by SDS-PAGE. Elution profile results (SDS-gel) will be sent to customer for decisions on pooling of fractions. Buffer exchange and concentration for additional charge (see below).

Delivered: Purified protein in elution buffer with final amount determined by efficiency of binding and elution to octyl- or butyl Sepharose.
Time: 4-6 days.