Koch Lab:Protocols/Kinesin/DLS Kinesin Aggregation

Intro

In a publication from Bachand Lab at Sandia, we studied ATP-inhibition of kinesin aggregation. See: Koch_Lab:Publications#Kinesin_.2F_Microtubule_Molecular_Motor_System figure and link to publication. We did this using dynamic light scattering as a qualitative measure of aggregation.

Instrument

(Details to come -- we used a commercial 90 degree temperature-controlled DLS system.)

Method

Cleaning DLS Cuvette

• I wear gloves to prevent fingerprints on the cuvette.
• Squirt some detergent / water mixture into the cuvette.
• Rinse liberally with water. I normally use nanopure water straight from the “tap” and will rinse the cuvette about 10 times, vigorously shaking the water out after each rinse. The point is to dilute out all the detergent and protein that was in there.
  • Careful not to drop the cuvette!
• Use pressurized nitrogen (in the hood) to blow dry the cuvette.

Preparing Sample

• A protein concentration of about 0.2 mg / ml is a good concentration. For example, dilute stock protein of 2 mg / ml 1:10 into final DLS buffer
  • I make about a 70 ul sample
• Mix sample in a microfuge tube, then spin for 1 minute at the maximum rate in the +4C microfuge. (This is roughly 20,000 g for 1 minute.)
  • The purpose of this step is to get rid of microscopic (and macroscopic for that matter) air bubbles, which will wreak havoc on the DLS data
• VERY GENTLY pipette the solution (about 65 ul) into the bottom of the cuvette.
  • Take care not to introduce dust or air bubbles!

Taking DLS Data

(Details to come -- email Steve to ask, and also see above link to Fungal Genetics paper.)