IMAC Application Note:
Syringe Columns for His-GFP Purification
Introduction
Cloning, expressing and purifying recombinant proteins are a cornerstone for many molecular biology and biochemistry labs. Recombinant proteins are an essential reagent yet preparations remain a tedious process. These small volume protein purifications remain a bottleneck to life science research. New tools are needed to simplify the procedures, increase throughput, and drive down costs. Gravity columns offer users the most flexibility but are inconvenient to pack and maintain. These columns should not be allowed to run dry so the user is at the mercy of watching the columns drip. Magnetic beads are a popular choice for purification of antibodies but are limited by capacity and the incubation time for a binding process that relies upon Brownian Motion. Furthermore, magnetic beads are a single use system, which drives up the cost. Spin columns are convenient and easy to use but sacrifice performance. And the single use design of spin columns are also an expensive choice. Here, syringe columns are presented as a suitable choice for performance and speed with the benefit of being reusable. Syringe columns are a suitable alternative to plate-based, gravity, magnetic bead and FPLC applications because of intuitive procedures. These syringe columns purify samples quickly without sacrificing performance. In addition, syringe columns are reusable making them very cost effective.
Materials and Methods
His-tagged GFP was expressed in E. coli transformed with plasmid driven by a Lac promotor. The expression was induced by IPTG for 6 hours. Cells were harvested by centrifugation, resuspended in resuspension buffer and lysed with Bug Buster. Lysate was clarified by centrifugation. Samples were prepared by diluting 250 µL of lysate with 750 µL TBS (Quality Biological, 35058-101). 1 mL sample was processed through the 5 mL syringe column packed with 100 µL of Ni-Immobilized Metal Affinity Chromatography (Ni-IMAC) resin. The syringe column was fitted with a luer lock needle and was equilibrated by aspirating and dispensing 1 mL of TBS. After equilibration, 200 µL of air was used to blow out the interstitial space. The column captured the His-GFP from the 1 mg/mL sample using 8-16 cycles of aspirate and dispense. After the final dispense, 200 µL of air was used to blow out the syringe column. The syringe column was washed with 1 mL 5 mM imidazole using 2 aspirate and dispense cycles. This was followed by 2 aspirate and dispense cycles in 10 mM imidazole. After the last blow out, 500 µL of air was aspirated and dispensed from the syringe column. The His-GFP was eluted in two sequential fractions of 400 µL 250 mM imidazole.
The eluted His-GFP was analyzed by the NanoDrop OneC (Thermo Fisher Scientific, ND-ONE-W).
Results
High efficient binding and recovery of His-GFP using Syringe Columns
The flow through of the sample was monitored using the NanoDrop after 8, 12 and 16 aspirate and dispense cycles. The majority of the His-GFP was recovered in elution 1 (Table 1). Elution 2 recovered about 1.2 mg of protein for a total of almost 7 mg of His-GFP.
Discussion
Syringe columns offer an attractive choice for routine protein purification. The syringe-based format is intuitive and easy to use. These offer high performance and are cost effective. Almose 7 mg of His-GFP was recovered in about 5 minutes.