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| Product Name | Colloidal Silica Density Gradient Medium |
| Catalog No. | RESK-0253 |
| Description | This product is a versatile and easy-to-use low-density gradient separation medium for the isolation of cells, subcellular particles, bacteria and viruses, and even for separating damaged cells and debris from intact viable cells. It consists of silica particles coated with a monolayer of polyvinylpyrrolidone (PVP), with a particle diameter of 15–30 nm and a free PVP content of only 1–2%. Due to the heterogeneity of particle size, the particles sediment at different rates during centrifugation, spontaneously generating a homogeneous, isotonic gradient with densities ranging from 1.0 to 1.3 g/mL. Most biological particles with sedimentation coefficients greater than 60S can be separated with this medium. |
| Intended Use | Density gradient isolation of cells, subcellular components, membrane vesicles, bacteria and viruses; separation of damaged cells and debris from intact viable cells; also commonly used for isolation and purification of plant protoplasts, chloroplasts and mitochondria. For research use only. |
| Principle / Technology | Colloidal silica particles (15–30 nm) coated with PVP sediment at different rates during centrifugation due to their size heterogeneity, spontaneously forming a homogeneous, isotonic density gradient (1.0–1.3 g/mL). Biological particles band at positions corresponding to their buoyant densities, enabling gentle separation under iso-osmotic conditions. |
| Sample Type | Cells (including sensitive cells), subcellular particles, organelles, membrane vesicles, bacteria, viruses, plant protoplasts, chloroplasts, mitochondria |
| Performance Range / Specifications | Sterile solution, colorless to pale yellow Density of undiluted medium: 1.135 g/mL (1.130 ± 0.005 g/mL) Gradient density range: 1.0–1.3 g/mL, isotonic throughout the gradient Viscosity: 10 ± 5 cP at 20 °C Particle diameter: 15–30 nm Free PVP content: 1–2% Suitable for particles with sedimentation coefficients >60S (down to ~70S for larger viruses) |
| Specificity | Non-ionic, chemically inert, non-cytotoxic; does not adhere to cell membranes. |
| Reaction Conditions / Protocol | 1. Preparation of isotonic working solution (SIP) Mix 9 volumes of the undiluted medium (v/v) with 1 volume of 1.5 M NaCl, 10x concentrated culture medium, 1.5 M PBS or 2.5 M sucrose solution. Adjust osmolarity with salt or distilled water to the desired final value. Since cell density depends on osmolarity, the osmolarity of the SIP solution should routinely be adjusted with an osmometer to ensure reproducibility. For SIP in saline, density = 1.123 g/mL; for SIP in sucrose, density = 1.149 g/mL (assuming stock density of 1.130 g/mL). 2. Dilution of SIP to lower densities Dilute SIP directly with 0.15 M NaCl or 1x cell culture medium (normal osmolarity) for cell separation, or with 0.25 M sucrose for subcellular or viral separation. (Density of 0.15 M NaCl is ~1.0046 g/mL; density of 0.25 M sucrose is ~1.032 g/mL.) Example: to dilute 55 mL of SIP to a final density of 1.07 g/mL, 44.6 mL of 0.15 M NaCl is required. Actual density should be verified with a densitometer or refractometer. 3. One-step dilution to a desired density (optional) In a graduated cylinder, add 1.5 M NaCl or 2.5 M sucrose to 1/10 of the final volume, add the calculated volume of stock medium, and make up to the final volume with distilled water. Example: to prepare 100 mL of working solution at density 1.07 g/mL in 0.15 M NaCl, add 49.4 mL of stock medium (density 1.130 g/mL) to 10 mL of 1.5 M NaCl and make up to 100 mL with distilled water. 4. Preparation of discontinuous density gradients Dilute SIP to a series of desired densities; pre-wet the tube wall with bovine serum to obtain sharp interfaces; layer the solutions from highest to lowest density along the tube wall using a pipette or a syringe with a wide-bore needle. 5. Sample loading and centrifugation Apply a moderate sample volume and cell concentration; centrifuge generally at 400 × g for 20–25 min with slow, smooth acceleration and deceleration. Collect cells at the interfaces or within layers as appropriate. Preformed gradients allow separation at low centrifugal force (200–1000 × g) within minutes to tens of minutes. 6. Washing The medium is non-cytotoxic and does not adhere to cell membranes, so removal is generally unnecessary; cells can be transferred directly to culture, viruses used directly for infection, and organelles used directly for metabolic studies. If removal is desired: wash cells 2–3 times with saline or PBS at a 5:1 volume ratio, centrifuging at 200 × g for 2–10 min each time; for viruses or subcellular particles, pellet the silica particles by high-speed centrifugation (100,000 × g, 2 h in a swing-out rotor or 90 min in a fixed-angle rotor) and retain the supernatant. |
| Components / Formulation | Colloidal silica particles coated with polyvinylpyrrolidone (PVP); particle diameter 15–30 nm; free PVP content 1–2%. Non-ionic. |
| Storage Conditions | Shipped at room temperature. Store unopened at 4–30 °C. After opening, store aseptically at 2–8 °C. If stored under non-aseptic conditions, freeze at -20 °C (leaving sufficient headspace for expansion) to prevent microbial growth. Frozen solutions may form a gradient upon thawing and must be mixed thoroughly before use. |
| Shelf Life | Unopened: 5 years at room temperature; after opening: at least 2 years when stored aseptically at room temperature, or up to 6 months at -20 °C under non-aseptic conditions. |
| Package Specifications | 100 mL |
| Product Form | Sterile liquid, colorless to pale yellow |
| Quality Control | Sterile solution; density 1.135 g/mL; viscosity 10 ± 5 cP at 20 °C |
| Purity | Free PVP content only 1–2% |
| Concentration | Undiluted stock density: 1.135 g/mL (1.130 ± 0.005 g/mL) |
| pH Range / Optimal pH | Can be adjusted to physiological ionic strength and pH |
| Shipping Conditions | Shipped at room temperature. |
| Expiration Date / Stability | Unopened product is stable for 5 years at room temperature; opened product is stable for at least 2 years when stored aseptically at room temperature; up to 6 months at -20 °C under non-aseptic conditions. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Compatible with cells, subcellular particles, membrane vesicles, bacteria and viruses; gentle and non-toxic; does not adhere to cell membranes. Polycarbonate (PC) centrifuge tubes are recommended. |
| Recommended Buffer System | Best diluted in buffer, physiological saline or 0.25 M sucrose solution. For cell samples, buffers such as PBS can be used; for subcellular particles, which may aggregate in the presence of salt ions, 0.25 M sucrose solution is recommended. |
| Application Notes / Precautions | Adjust osmolarity with an osmometer for reproducible results, as cell buoyant density depends on osmolarity. Densities calculated from formulas are approximations; verify actual density with a densitometer or refractometer. Use slow, smooth centrifuge acceleration/deceleration for discontinuous gradients. Avoid excessive sample volume or cell concentration, which can impair separation and recovery. The undiluted medium can be re-autoclaved (120 °C, 30 min). |
| Heat Inactivation | The undiluted medium can be re-autoclaved at 120 °C for 30 min. |
For research use only, not for clinical use.
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