We present two efficient protein purification strategies using our AcroPrep™ Advance 96-well filter plates. These methods enable rapid, small-scale separation of protein mixtures using either packed chromatography resins or ready-to-use Mustang™ ion exchange membranes. The first approach combines Supor™ membranes with multimodal resins for batch-mode purification. The second uses Mustang™ Q and S membranes to achieve high-resolution, charge-based separation. Both methods support high-throughput screening, minimize sample consumption, and can be scaled for downstream applications. This flexible platform helps you streamline purification workflows and accelerate method development.
Introduction
The introduction of 96-well filter plates combined with chromatography media has emerged as an efficient tool for the fractionation of small-volume protein samples. We can use this format to develop protein purification strategies or as a platform for moderate to high-throughput protein isolation. In either case, the purified sample can be used for further analysis and downstream applications.
In this article, we review the development of two methods for protein purification strategies using small quantities of protein mixtures:
- One method uses AcroPrep™ Advance filter plates with Supor™ membrane combined with a mixed-mode (multimodal, MM) chromatography resin to yield complete separation of three proteins in a protein sample.
- Another method employs AcroPrep™ Advance filter plates with Mustang™ ion exchange chromatography (IEX) membrane to separate a mixture of three different proteins.
Utilizing a filter plate-based strategy allows us to rapidly screen multiple purification schemes. Fractionation and protein purification methods developed within the plate can be used to optimize larger-scale purification.
High-throughput screening on 96-well plates
Our AcroPrep™ Advance 96-well filter plates are optimal for analytical applications. By packing chromatography resins in the wells of the plates, we can use them for protein or nucleic acid purification. One advantage of using our filter plates is their capability to quickly screen various conditions, allowing optimization of the chromatography resin and purification conditions with minimal sample consumption. This rapid screening ability and process condition optimization can be transferred and confirmed or scaled up.
The flexible 96-well format of our AcroPrep™ Advance filter plates can be used with both liquid-handling robotic systems or manual multichannel pipettes. Sample or buffer recovery can be conducted using vacuum aspiration or centrifugation to draw the liquid through the membrane and bead bed into a 96-well receiver plate.
We performed the screening of multiple conditions to optimize the purification step with an AcroPrep™ Advance 96-well filter plate. Once we had equilibrated the selected resin and resuspended it as a 50% slurry in the equilibration buffer, we dispensed the desired amount into the wells to a final volume of 50 μL per well. After dispensing the slurry into the wells of the plate, we then aspirated the equilibration buffer using a multiwell plate vacuum manifold.
Next, we performed a sequence mimicking a chromatographic run on the plates. For each step of the sequence, we pipetted the corresponding solution into the wells. Once the wells were filled, we covered the filter plate with sealing tape and incubated while shaking.
After incubation, we drained the liquid from the wells using the vacuum manifold and collected it in a 96-well receiver plate. We then analyzed these individual fractions by HPLC, ELISA, or other analytical methods. By using this data, we determined the set of parameters providing the optimal selectivity for our application—whether it be protein concentration and recovery or contaminant removal.
We can pack the chromatography resin in two ways (Fig 1), following manufacturing recommendations.
Fig 1. Flowchart showing the methods of packing a chromatography resin in the filter plate.
Small-scale protein purification using packed chromatography resins
An AcroPrep™ Advance filter plate with Supor™ 0.45 or 1.2 μm membrane (depending on the resin wet-bead size), can be packed with various chromatography resins for small-scale, batch-mode protein purification studies.
This technique can be applied using a variety of sample types and optimized with the chromatography resin suited to meet your purification requirements. You should develop purification strategies based on known physical and chemical characteristics of the target molecule, such as net charge, hydrophobicity, and affinity for metals or ligands. You can then screen chromatography resins and variations in pH and conductivity to determine the ideal candidates for the purification of our target molecules.
Membrane-based protein purification
Membrane chromatography was created to address mass transfer limitations of conventional resin-based methods, enabling improved flow distribution, faster rates, and more efficient protein purification or contaminant removal. These attributes translate into higher throughputs and reduced processing times.
We offer filter plates with two membranes with strong IEX ligands: quaternary amine and sulfonic functional groups on Mustang™ Q (strong anion exchange) and Mustang™ S (strong cation exchange) membranes respectively. These AcroPrep™ Advance 96-well filter plates are optimal for small scale, primary protein purification applications.
If resin packing is inappropriate for your application, you can consider ion exchange with AcroPrep™ Advance 96-well filter plates with Mustang™ IEX chromatography membranes instead.
Mustang™ Q anion membrane performance
We can show the performance of Mustang™ Q membrane by separating a three-protein mixture using a salt elution strategy. SDS-PAGE analysis of fractions (Fig 2) shows a clear separation of each protein.
- Cytochrome C (Mr 12 000) does not bind to the Mustang™ Q membrane at pH 8.8 (Lane FT)
- Conalbumin (Mr 78 000) is eluted with the addition of 0.1 M NaCl (Lane E1)
- Albumin (Mr 67 000) is eluted with 0.4 M NaCl (Lane E2)
Fig 2. Complete separation of proteins with AcroPrep™ Advance filter plates with Mustang™ Q chromatography membrane. 1.25 to 3.75 μg of total protein (not reduced) loaded onto 12% SDS-PAGE. Lanes: MW = molecular weight markers; Load = protein mixture; FT = flowthrough; E1 and E2 = 0.1 and 0.4 M eluate fractions. GelCode stain used.
Mustang™ S cation membrane performance
We saw similar levels of protein separation with Mustang™ S membrane in AcroPrep™ Advance filter plates, as seen by SDS-PAGE analysis of these fractions (Fig 3).
- All three tested proteins bind to the Mustang™ S membrane in Na Acetate, pH 4.5
- Trypsinogen (Mr 24 000) elutes first with 0.2 M NaCl (Lane E1)
- Cytochrome C (Mr 12 000) requires higher salt and elutes with 0.5 M NaCl (Lane E2)
- Lysozyme (Mr 14 400) requires 1.0 M NaCl for elution (Lane E3)
AcroPrep™ Advance filter plates with Mustang™ IEX membranes have demonstrated robust charge-based protein separations allowing for rapid protein purification.
Fig 3. Complete separation of proteins with AcroPrep™ Advance filter plates with Mustang™ S membrane: 1.25 to 3.75 μg of total protein (not reduced) loaded onto 12% SDS-PAGE. Lanes: MW = molecular weight markers; Load = protein mixture; FT = flowthrough; E1 and E2 = 0.1 and 0.4 M eluate fractions. GelCode stain used.
Discussing advantages of using Mustang™ IEX membranes
AcroPrep™ Advance filter plates with Mustang™ IEX membranes have several performance advantages over resin-based IEX purification. Increased flow rates and better flow distribution allow for rapid binding and elution of biomolecules and wash steps are immediate. Large membrane pores increase accessibility to the IEX chemistries, either for purification or contaminant removal.
In addition, our membranes have reduced hold-up compared to the resin. This increases wash efficiency and yields more concentrated elutes. These factors combine to create an efficient platform for protein purification. When reviewing protein purification strategies using IEX chemistries, we highly recommended that you analyze the performance of the membrane chromatography.
Conclusions
In this article, we demonstrated two methods for fractionation of protein samples using AcroPrep™ Advance filter plates.
For the first method, we used an AcroPrep™ Advance filter plate (fitted with a 1.2 μm Supor™ membrane) and combined it with chromatography resin for small scale batch-mode protein separations. Using MM resins, capable of hydrophobic and IEX interactions, we separated a three-protein mixture. This technique can be applied to various sample types and optimized with the ideal chromatography resin for your purification requirements.
Develop your purification strategies based on the physical and chemical characteristics of the target molecule, such as net charge, hydrophobicity, and affinity for metals or ligands. Screen IEX, MM, and affinity chromatography (AC) resins to find the best candidates for your target molecule's purification.
We achieved the second fractionation method of additional three-protein mixtures by using Mustang™ Q and S membranes. In both cases, we bound proteins to the membrane and eluted them with salt. Each fraction contained one protein that could be used for downstream applications. Unlike filter plates combined with chromatography resin, AcroPrep™ Advance filter plates with Mustang™ membrane are ready-to-use separation units.