Protein Purification by Gel filtration Chromatography (CAT#: STEM-MB-1297-LGZ)

The principle is to use the difference of protein molecular weight or molecular shape to separate. When the sample moves down from the top of the column, large protein molecules cannot enter the gel particles and are quickly eluted; smaller protein molecules can enter the gel particles, and the retention time of the proteins entering the gel is also different in the gel. The larger the molecular weight, the earlier the outflow time, and finally separate proteins with different molecular sizes.
Generally, most gel matrices are prepared by chemically cross-linking polymer molecules, and the degree of cross-linking determines the pore size of the gel particles. Commonly used chromatography matrices: Sephadex, Sepharose, Bio-Gel P, etc. Highly cross-linked matrices can be used to separate proteins and other smaller molecules, or to remove low-molecular-weight buffer components and salts, while larger-pore gels can be used to separate protein molecules. The choice of a gel with a suitable pore size depends largely on the molecular weight of the target protein and the molecular weight of the foreign protein.

For large molecules, macromolecular complexes purification.

1. Gel sterilization treatment: After washing the column with ultrapure water, wash the column forward with 0.5mol/L NaOH at a flow rate of 3mL/min for 3 column volumes.
2. Equilibration: After NaOH treatment, wash 2 column volumes with ultrapure water, then wash 5~10 column volumes with 7.0PH buffer solution containing 200mmol/L NaCl and 20mmol/L PB.
3. Sample loading: After equilibration, select the sample pump to load the sample, the sample loading flow rate is 3mL/min, and the sample volume is 1mL.
4. Elution: After loading the sample, use the equilibration buffer for elution.
5. Cleaning and preservation.
After purification, back wash 2 column volumes with 0.5mol/L NaOH for 30-60 minutes. After washing, forward wash 5 column volumes with ultrapure water, then wash 3 column volumes with 20% ethanol, then remove the column, seal both ends, and store at low temperature.

• Chromatography medium
• Chromatographic column
• Chromatography equipment
• Mixed sample
• NaOH 0.5 mol/L
• NaCl 200mmol/L
• PB 20mmol/L
• PH7.0 buffer