Red Sepharose 6FF is an affinity filler prepared by immobilizing Reactive Red 120 (also known as Procion Red HE-3B) reactive dye onto agarose microspheres. Reactive Red 120 is a polycyclic dye with structural similarity to NADP+ , and can be used for purification of nucleotide-dependent enzymes, lipoproteins, lactate dehydrogenase, plasminogen, peptides, hormones, etc. In addition to its use as an affinity filler, the ligand of this product contains aromatic rings and anionic groups, enabling non-affinity purification through electrostatic or hydrophobic interactions.
Aladdin Red Sepharose 6FF is supplied in 20% ethanol, with a gel to preservative solution volume ratio of 1:1. The product specification refers to the actual gel volume.
Parameter | Specification | Matrix | 6% Cross-linked Agarose | Ligand | Reactive Red 120, ~2 μmol/mL | Particle Size Range ① | 45–165 μm | Mean Particle Size | ~90 μm | Dynamic Binding Capacity ② | ~15 mg BSA/mL | Recommended Operating Flow Rate | 60–300 cm/h | Maximum Flow Rate & Pressure ③ | 1500 cm/h, 0.5 MPa | Working pH | 4–8.5 (recommended); 3–12 (long-term stable); 2–14 (short-term stable) | Chemical Stability | Stable in common aqueous buffers, 0.5 mol/L NaOH, 8 mol/L urea, 6 mol/L guanidine hydrochloride, 70% ethanol | Storage | 20% ethanol, 2–8℃ | Shelf Life | 5 years |
|
Notes:
①More than 90% of microspheres by volume are within this range.
② DBC10% tested at 10% breakthrough, 6 min residence time.
③ Maximum tested flow rate at 10 cm bed height.
Instructions for Use
1. Column Packing
The following describes column packing procedures for connection to a chromatography system.
(1)Equilibrate all materials to the temperature of chromatography; degas liquids if possible.
(2)Calculate filler volume: Settled filler volume = Column volume × Compression factor. Settled volume is the stable volume after full sedimentation of the filler in 20% ethanol storage solution. The compression factor of Red Sepharose 6FF is 1.15. Achieve target compression by lowering the top adapter or high-flow packing.
(3)Wash filler: Mix the filler suspension thoroughly, measure the required volume, remove liquid by vacuum filtration, and wash with ~3 column volumes of purified water (repeat 3 times) to remove storage solution.
(4)Prepare packing suspension: Transfer filler to a suitable container, add appropriate packing buffer to make 50–75 v% suspension, and mix well before use.
(5)Pre-packing preparation: Add packing buffer to the bottom of the cleaned column to eliminate air from the lower frit and column base; leave a small volume of packing buffer, tighten the lower end cap, and vertically align the column.
(6)Packing: Slowly pour the well-mixed filler suspension into the column in one portion (use a packing reservoir if needed), running it down the inner wall to avoid bubbles. After adding all filler, fill the packing reservoir with packing buffer, fasten the top cap, and connect the column to the system.
(7)Compaction: Let filler settle naturally (or at low flow rate 50–150 cm/h) until the bed–liquid interface is clear. Remove the packing reservoir, install the top adapter, and lower the adapter to the interface. Continue compaction at high flow rate (see table; keep pressure <0.3 MPa) until the interface is stable; mark the bed height. Stop the pump, open the adapter vent/cap, close the column bottom valve/cap, lower the adapter to the position corresponding to the compression factor below the mark, and tighten. Equilibrate the packed bed at high flow rate.
Packing Condition | Red Sepharose 6FF | Compression Factor | 1.15 | Packing Flow Rate | 600 cm/h |
|
2. Column Efficiency Measurement & Evaluation
Column efficiency can be tested after packing and before use to verify quality, using HETP (Height Equivalent to a Theoretical Plate) and asymmetry factor (As).Test with acetone or NaCl solution as sample, according to the table below.
Sample | 1.0% Acetone | 0.8–1.0 M NaCl | Sample Volume | 1.0% of column volume | 1.0% of column volume | Mobile Phase | Pure Water | 0.4 M NaCl | Flow Rate | 30 cm/h | 30 cm/h | Detector | UV-280 nm | Conductivity |
|
Calculate HETP, theoretical plate number (N), and asymmetry factor (As) from UV or conductivity profiles:
HETP = L / N
N = 5.54 × (VR/ Wh)2
As = a / b
Where:L = bed height; VR = retention volume; Wh = peak width at half height;a = first half-width at 10% peak height; b = second half-width at 10% peak height.
General criteria: HETP < 3 × mean particle size (HETP/D₅₀ < 3); As = 0.8–1.5.
3. Equilibration & Loading
Equilibrate the column with Buffer A at operating flow rate before loading, typically 5–10 column volumes until conductivity, pH, and other signals are constant and match Buffer A. Common buffers such as phosphate or Tris-HCl are suitable.
Sample loading can be set as mg target protein per mL filler, usually 50–80% of DBC10%. For this medium (DBC10%~15 mg/mL), load 7.5–12 mg/mL. Alternatively, determine loading by target protein in flow-through.
Centrifuge samples (≥10000 g) and filter (0.22 or 0.45 μm) before loading to prevent clogging.After loading, re-equilibrate with 3–10 column volumes of Buffer A.
4. Elution
Target protein is typically eluted with Buffer A supplemented with 3 M NaCl or 2 M KCl.
5. Regeneration
After purification, regenerate the column by alternating washes with weakly acidic and weakly basic buffers 3–4 cycles, e.g., 0.1 mol/L NaAc + 0.5 mol/L NaCl (pH 4.5) and 0.1 mol/L Tris·HCl + 0.5 mol/L NaCl (pH 8.5). Re-equilibrate with equilibration buffer afterward.
6. Cleaning-in-Place (CIP)
Standard elution and regeneration usually suffice for thorough cleaning. For increased backpressure or cross-contamination prevention, use: 0.1–0.5 mol/L NaOH, 70% ethanol, or 30% isopropanol. CIP effectively removes impurities; reverse-flow cleaning is more efficient. After CIP, wash with 3–5 column volumes of pure water to remove cleaning agents.
CIP Procedure | Purpose | 0.1–0.5 mol/L NaOH, ~0.5 h contact time (longer for endotoxin removal); Buffer A, 5–10 CV; Water, 3–5 CV | Remove protein precipitates, hydrophobic proteins, pyrogens, etc. | 30% Isopropanol or 70% ethanol, 5–10 CV; Water, 3–5 CV | Remove tightly bound lipoproteins, liposomes, etc. |
|
7. Storage
The medium is supplied in 20% ethanol. After use, store in 20% ethanol with 0.5 M NaCl added. Recommended storage temperature: 2–8℃. Do not freeze.
ZJ26F0636577