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

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Sucrose Gradient Centrifugation (Velocity Sedimentation) Protocol for Macromolecules such as Proteins

Purpose: To separate out different sizes of macromolecules such as proteins; can be used as a form of protein purification

Gradient Preparation

· Use the P1000 to pipet 500uL 20% sucrose solution onto the bottom of each thin-walled tube
· Use the P200 to pipet 500uL of each of subsequent concentrations of sucrose (15, 10, 5%)
· Pipet slowly, putting pipet tip, just slightly below the liquid and slowly moving it up
· Leave for 1 hour at room temperature, lid on, but slightly open

· Leave on ice for 10 minutes to 30 minutes afterwards

Sample (e.g. Protein) Preparation

· Spin down 5-10 minutes 100,000 x g
· Load 100-200uL of sample slowly onto the sucrose gradient
· Balance the tubes

· Spin for the desired amount of time in swing-out buckets

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