Native Protein Purification Service

We accept native protein purification samples from various sources and separates and purifies the target proteins required by clients. Sample forms include animal and plant tissues, cells, microbial cells, supernatants from lysed microbes, denatured solutions, secretion/expression fluids, and powders.

The first step in isolating and purifying the target protein is to release the protein from its original sample in a dissolved state while maintaining its native conformation and biological activity. We select appropriate sample pretreatment methods according to the sample form.

For animal materials, connective tissues and fat are removed first. For seeds, the shells or seed coats are removed to avoid contamination by substances such as tannins. Oilseeds are preferably defatted with low-boiling organic solvents such as ether. Then, depending on the sample, suitable methods are chosen to break down tissues and cells.

Animal tissues and cells can be disrupted by electric homogenizers, blenders, or ultrasonic treatment. Plant tissues and cells usually require grinding with quartz sand or glass powder along with an appropriate extraction buffer, or treatment with cellulase. Common methods to disrupt bacterial cell walls include ultrasonic disruption, sand grinding, high-pressure extrusion, or lysozyme treatment.

After tissue and cell disruption, proteins are extracted using appropriate buffers. Insoluble materials such as cell debris are removed by centrifugation or filtration.

If the target protein is mainly concentrated in specific cellular components, such as nuclei, chromosomes, ribosomes, or soluble cytoplasm, differential centrifugation can separate these components, allowing collection of the fraction as the starting material for further purification. If the target protein is bound to membranes or membranous organelles, membrane structures must be disrupted by ultrasound or detergents, followed by extraction using suitable media.

Based on the sample type and the properties of the target protein, native protein purification generally falls into three categories: Purification of Native Macromolecular Proteins, Purification of Native Small-Molecule Proteins, and Purification of Mixed or Unknown Protein Samples.

Purification of Native Macromolecular Proteins: According to the physicochemical properties of the target protein, the process typically begins with various ion exchange chromatography steps. Different buffer systems are explored, and appropriate pore size of size exclusion chromatography columns is selected for separation and purification.

Purification of Native Small-Molecule Proteins: Native small-molecule proteins usually refer to various natural enzymatic hydrolysates obtained through enzymatic digestion, with molecular weights typically below 10 kDa. Clients often need to isolate high-activity peptide sequences. Based on years of experience, we typically begin with multistage size exclusion chromatography (such as S200, S75, G15) to remove macromolecular impurities. The resulting small-molecule mixture is then further purified using macroporous resin or reversed-phase resin. Finally, the sequences of small peptides are identified using HPLC and MS/MS.

Purification of Mixed or Unknown Protein Samples: In some special cases, clients may only provide an activity assay without any information about the target protein. In such scenarios, we develop a Purification Plan based on the presumed characteristics of the target protein. The purified products are then validated through mass spectrometry, activity assays, and other approaches to ensure the acquisition of a high-purity, biologically active target protein.

Figure 2: Schematic diagram of ion-exchange chromatography

Figure 3: Schematic diagram of gel-filtration chromatography

We have established a comprehensive native protein purification system, offering a variety of purification methods, including affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, HPLC and others. Equipped with advanced AKTA Purification Systems and a wide range of pre-packed purification columns, we are capable of purifying native protein samples from various sources. With over a dozen types of GE purification media available, we can perform large-scale, high-throughput protein separation and purification to obtain high-purity proteins, significantly saving researchers valuable time and meeting diverse experimental needs.

Our native protein purification process is designed not only to select the most suitable purification techniques but also to optimize experimental parameters. Each purification step is efficiently connected to minimize steps, labor, and cost, enabling maximum recovery of target proteins from minimal sample input while preserving biological activity. This ensures that the purified proteins can be used for activity testing, experimental analysis, and downstream applications. Additionally, KMD provides fully traceable documentation to support customers in establishing their own purification processes.

Case 1: Purification of Cell Culture Additives

Figure 4. Purification Profile of XX Protein

Figure 5. SDS-PAGE Analysis of XX Protein Purification

Case 2: Purification of Pear Samples

Figure 6. Gel Filtration Chromatography of XX Protein

Figure 7. SDS-PAGE Analysis of Purified XX Protein