High-throughput protein purification is commonly performed on E. coli cultures expressing a fusion protein that are grown in 96-well deep-well plates. These small cultures (~1mL) are ideal for expressing a library of cDNAs or to generate a series of variants for a particular protein to perform mutational analysis. Because 96-well spin plates are compatible with automated liquid handling systems or properly equipped centrifuges, immobilized metal affinity chromatography (IMAC) and other fusion tag affinity resins can be integrated into high-throughput protein purification workflows.
In addition, using an optimized cell lysis reagent, such as Thermo Scientific B-PER Direct Bacterial Protein Extraction Kit (Part No. 90080), the cultured cells can be lysed directly in their growth media and applied to the filter plate affinity resin, eliminating the need to centrifuge and aspirate these types of cultures prior to processing.
The following Thermo Scientific Pierce 96-well Spin Plates are available for fusion protein purification:
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Protein Purification Technical Handbook
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HisPur Cobalt Spin Plates
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Thermo Scientific HisPur Cobalt Spin Plates use cobalt ions immobilized onto a 6% agarose resin with a tetradentate chelator and have been optimized for high-purity 6xHis-tagged protein purification. Immobilized cobalt exists as a divalent cation and associates with histidine amino acid side chains for the selective enrichment of His-tagged proteins. Elution is performed with an excess of imidazole.
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96-well Filter Spin Plate Specifications and Applications
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| Technical Specifications for Thermo Scientific HisPur Cobalt Spin Plates |
| Resin |
crosslinked 6% agarose |
| Ligand |
cobalt-charged tetradentate chelator |
| Resin bed volume |
100µL (400µL of 25% resin slurry) |
| Binding capacity |
1mg/well at >90% purity of a 28-kDa His-tagged protein from a bacterial source |
| Sample volume per well |
≤400µL |
| Assay time |
45 minutes |
| Metal ion capacity |
≥12µmol cobalt/mL resin |
| Coefficient of variation (CV %) of protein yield |
<10% |
| pH limits |
2 to 14 (2 hours); 3 to 10 (24 hours) |
| Centrifugation speed |
100 to 500 x g (maximum 700 x g); 3 minutes |
| Storage buffer |
ultrapure water with 20% ethanol |
| Storage temperature |
4°C |
| Suggested balance plate |
use Part No. 45205 |
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HisPur Cobalt Agarose Resins, Columns and Plates
96-well Filter Collection Plates
96-well Spin Balance Plate
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HisPur Ni-NTA Spin Plates
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Thermo Scientific HisPur Ni-NTA Spin Plates use nickel ions immobilized onto a 6% agarose resin with nitrilotriacetic acid (NTA) chelator and is the most commonly used IMAC resin due to its high binding capacity (60mg His-tagged protein/mL of resin).
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96-well Filter Spin Plate Specifications and Applications
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| Technical Specifications for Thermo Scientific HisPur Ni-NTA Spin Plates |
| Resin |
crosslinked 6% agarose |
| Ligand |
chelated nickel |
| Resin bed volume |
50µL (200µL of a 25% slurry) |
| Binding capacity |
≥1mg of a 28-kDa 6xHis-tagged protein per well |
| Sample volume per well |
25 to 200µL |
| Assay time |
45 minutes |
| Metal ion capacity |
≥15µmol cobalt/mL resin |
| Coefficient of variation (CV %) of protein yield |
<10% |
| Centrifugation speed |
1000 x g |
| Storage buffer |
ultrapure water with 20% ethanol |
| Storage temperature |
4°C |
| Suggested balance plate |
use Part No. 45205 |
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HisPur Ni-NTA Agarose Resins, Columns and Plates
96-well Filter Collection Plates
96-well Spin Balance Plate
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Purification of 6xHis-tagged GFP from bacterial lysate using Thermo Scientific HisPur Ni-NTA Spin Plate. Bacterial lysate (0.45mg total protein) containing over-expressed recombinant 6xHis-tagged GFP was prepared in B-PER Bacterial Protein Extraction Reagent (Part No. 78243) and applied to each well of a Ni-NTA 96-well Spin Plate. The lysate, flow-through, wash and elution fractions were visualized on an SDS-PAGE gel stained with GelCode Blue Stain Reagent (Part No. 24590). The purity of the samples in lanes 6 to 8 was 90%, as determined by densitometry. M = MW marker, L = lysate, F = flow-through, W = wash, E = eluate. |
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Pierce Glutathione Spin Plates
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Thermo Scientific Pierce Glutathione Spin Plates use a glutathione tripeptide (GSH) immobilized on a 6% crosslinked agarose. The GSH ligand is specific for glutathione-S-transferase (GST), and the immobilized glutathione agarose used in these spin plates can purify up to 10mg of GST-fusion protein per milliliter of resin.
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96-well Filter Spin Plate Specifications and Applications
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| Technical Specifications for Thermo Scientific Pierce Glutathione Spin Plates |
| Resin |
crosslinked 6% agarose |
| Ligand |
reduced glutathione tripeptide (GSH) |
| Resin bed volume |
50µL (100µL of a 50% slurry) |
| Binding capacity |
≥1mg recombinant glutathione-S-transferase (GST) per well. |
| Sample volume per well |
25 to 200µL |
| Assay time |
45 minutes |
| Coefficient of variation (CV %) of protein yield |
<10% |
| Centrifugation speed |
1000 x g |
| Storage buffer |
ultrapure water with 0.05% sodium azide |
| Storage temperature |
4°C |
| Suggested balance plate |
use Part No. 45205 |
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Pierce Glutathione Agarose Resins, Columns and Plates
96-well Filter Collection Plates
96-well Spin Balance Plate
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Purification of GST-labeled protease from HRV3C lysate using Thermo Scientific Pierce Glutathione Spin Plates. Bacterial lysate (1mg total protein) containing over-expressed recombinant GST-tagged HRV3C protease was prepared in B-PER Bacterial Protein Extraction Reagent (Part No. 78243) and applied to each well. The lysate, flow-through, wash and elution fractions were visualized on an SDS-PAGE gel stained with GelCode Blue Stain Reagent (Part No. 24590). The purity of the samples in lanes 6 to 8 was determined to be 90% by densitometry. M = MW marker, L = lysate, F = flow-through, W = wash, E = eluate. |
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