Sucrose Gradient Centrifugation (Velocity Sedimentation) Protocol for Macromolecules such as Proteins

Objective: To separate the different sizes of macromolecules such as proteins; can be used as a form of purification of proteins

Gradient Preparation

Use P1000 to Pipette 500uL 20% sucrose solution under any thin-walled tube
Use P200 to Pipette 500uL of each subsequent concentration of sucrose (15, 10, 5%)
Pipette slowly, placing the tip of the pipette, only slightly below the liquid and slowly move up
Let stand for 1 hour at room temperature, cover, but slightly open

Leave the ice for 10 minutes to 30 minutes after the

Samples (eg proteins) Preparation

5-10 minutes spun down 100,000 x g
Loading 100-200uL sample slowly to the sucrose gradient
balancing the tube

Rotate to the desired amount of time in a swing-out bucket

Carefully and slowly puncture holes (through styrofoam) right through the center of the tube in the needle 26.75cc (one attached to the 1mL syringe), collecting four-drop fractions, about 16 tubes Measuring concentration of sucrose in each tube with a refractometer (use 20uL) [normalize with normal first-butanol n-but should have a value of 1.397]