SUNRESIN
Stock Code 300487
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In the realm of biotechnology and molecular biology, purifying proteins with high specificity and yield is a cornerstone of research and industrial applications. One of the standout tools in this field is Ni Sepharose Excel, an advanced immobilized metal ion affinity chromatography (IMAC) resin designed to streamline the purification of histidine-tagged recombinant proteins. This blog post explores what Ni Sepharose Excel is, its unique properties, and its applications in protein purification, particularly for challenging samples like eukaryotic cell culture supernatants.
Ni Sepharose Excel is a nickel-charged affinity chromatography resin developed by Cytiva (formerly GE Healthcare Life Sciences). It consists of highly cross-linked 6% agarose beads (approximately 90 µm in size) coupled with a chelating ligand that is pre-charged with nickel ions (Ni²⁺). These nickel ions are exceptionally strongly bound to the ligand, resisting stripping even under harsh conditions, such as incubation with 10 mM EDTA or 1 M NaOH for 24 hours. This robust binding is what sets Ni Sepharose Excel apart from traditional IMAC resins, making it ideal for specific purification challenges.
The resin is designed primarily for the capture and purification of histidine-tagged (His-tagged) proteins, which are recombinant proteins engineered with a polyhistidine tag (typically 6–10 histidine residues). These tags bind specifically to the nickel ions on the resin, allowing for selective purification from complex mixtures.
High Resistance to Nickel Stripping: Unlike conventional IMAC resins, Ni Sepharose Excel’s nickel ions remain tightly bound even in the presence of stripping agents like EDTA. This eliminates the need for time-consuming sample pretreatment, such as dialysis or buffer exchange, which is often required to remove chelating agents from cell culture supernatants.
Direct Sample Loading: The resin supports direct loading of eukaryotic cell culture supernatants (e.g., from CHO or insect cells) and bacterial samples (e.g., E. coli), simplifying workflows and reducing protein degradation. This is particularly beneficial for secreted His-tagged proteins, which are often present in low concentrations in large sample volumes.
High Binding Capacity: Ni Sepharose Excel offers a binding capacity of up to 10 mg/mL of resin, ensuring efficient capture of target proteins even from dilute samples.
Versatile Formats: The resin is available in multiple formats, including loose resin (25 mL, 100 mL, 500 mL), prepacked HisTrap Excel columns (1 mL and 5 mL), and His Mag Sepharose Excel magnetic beads for small-scale purification and screening. This flexibility supports applications from early-stage research to large-scale production.
Compatibility with Large Sample Volumes: The flow properties of Ni Sepharose Excel make it suitable for purification at various scales, from manual methods to automated chromatography systems, allowing researchers to process large sample volumes efficiently.
Stability Across Conditions: The resin is compatible with a wide range of buffer conditions, including those containing reducing agents, denaturants (e.g., 8 M urea, 6 M Gua-HCl), and detergents, ensuring robustness during purification.
Ni Sepharose Excel is primarily used for purifying His-tagged proteins secreted into eukaryotic cell culture supernatants, such as those from Chinese Hamster Ovary (CHO) cells, insect cells (e.g., High Five™), or human embryonic kidney (HEK293) cells. Its ability to handle samples with stripping agents makes it a go-to choice for these applications. Key use cases include:
Purification of Secreted Proteins: Proteins secreted into eukaryotic cell culture media, such as human transferrin receptor 1 from BHK cells or the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, can be directly loaded onto Ni Sepharose Excel without extensive preprocessing, increasing yield and minimizing degradation.
Bacterial Protein Purification: While optimized for eukaryotic samples, the resin is also effective for purifying His-tagged proteins from bacterial systems like E. coli, such as the membrane protein insertase EcYidC for crystallization studies.
High-Throughput Screening: The His Mag Sepharose Excel format is ideal for small-scale experiments, enabling rapid capture of His-tagged proteins using magnetic devices during early-stage research.
Large-Scale Production: The resin’s flow properties and compatibility with prepacked columns (e.g., HisTrap Excel) make it suitable for scaling up purification processes in bioprocessing applications.
Remove Whole Cells: Centrifuge the sample to remove whole cells and prevent column clogging. For HisTrap Excel columns, no further clarification is needed, but for loose resin, filter the sample through a 0.45 µm filter to remove debris.
Adjust Buffer Conditions: Adjust the sample to match the binding buffer’s composition and pH (typically neutral, though pH 6.0 has been used successfully). Avoid using imidazole in the sample and binding buffer to optimize binding.
Column Preparation: If the column contains 20% ethanol (storage solution), wash with 5 column volumes of distilled water at a flow rate of 50–100 cm/h.
Equilibration: Equilibrate the column with at least 5 column volumes of binding buffer (e.g., 20 mM Tris-HCl, 500 mM NaCl, pH 7.4) at 150–600 cm/h.
Sample Loading: Load the sample at 150–600 cm/h. The resin’s design allows direct loading of large volumes without nickel stripping.
Washing: Wash with 20 column volumes of wash buffer (e.g., binding buffer with 20 mM imidazole) at 150 cm/h to remove unbound proteins.
Elution: Elute the His-tagged protein using an elution buffer (e.g., 20 mM Tris-HCl, 500 mM NaCl, 200 mM imidazole, pH 7.4). A one-step elution with 5 column volumes is often sufficient, but a linear gradient (10–20 column volumes) can improve purity at the cost of lower concentration.
Cleaning and Storage: Wash with 30% isopropanol or 0.1–0.5% nonionic detergent in 0.1 M acetic acid to remove hydrophobically bound contaminants. Store in 20% ethanol after washing with distilled water.
Imidazole Sensitivity: Some proteins may not bind if imidazole is present in the binding buffer. Test with lower (e.g., 0–10 mM) or no imidazole if binding issues occur.
Post-Purification Processing: If imidazole needs to be removed, use a desalting column. For further purification, size exclusion chromatography (SEC) can be employed, as seen in protocols for SARS-CoV-2 RBD purification.
Compared to other IMAC resins like Ni Sepharose 6 Fast Flow or Ni Sepharose High Performance, Ni Sepharose Excel excels in handling samples that cause nickel stripping. For instance, a study comparing HisTrap Excel (prepacked with Ni Sepharose Excel) and HisTrap FF crude (prepacked with Ni Sepharose 6 Fast Flow) showed that HisTrap Excel yielded 12.7 mg of total protein, including 8.9 mg of the target His-tagged hemagglutinin, while the yield from HisTrap FF crude was too low to quantify. Additionally, HisTrap Excel retained its nickel ions (visible as a blue color), whereas HisTrap FF crude did not.
Ni Sepharose Excel is a game-changer for researchers purifying His-tagged proteins, particularly those secreted into eukaryotic cell culture supernatants. Its ability to resist nickel stripping, support direct sample loading, and maintain high binding capacity (up to 10 mg/mL resin) simplifies workflows, increases yields, and preserves protein integrity. Whether you’re working on small-scale screening or large-scale bioprocessing, Ni Sepharose Excel offers unmatched versatility and efficiency, making it ideal for applications from structural biology to therapeutic protein production. For researchers seeking alternative resin-based solutions for protein purification, Sunresin Life Sciences offers a range of innovative chromatography resins that may complement or provide alternatives to IMAC-based purification, supporting diverse bioprocessing needs with high-performance materials. By leveraging robust purification technologies, scientists can confidently focus on downstream applications with reliable results.
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