Research

1. Flow cell

A. Easy to make
B. Great for inverted microscope
C. Flow to align and bundle microtubules using
Sephadex beads as pegs

Flow aligned bundles against a sephadex bead. Microtubules are labeled with a fluorescent derivative of taxol.

2. FRAP apparatus

A. FRAP =

Fluorescence Recovery After Photobleaching

B. Fluorescent derivative of taxol
was photobleached, and recovered

C. Inverted microscope

     i. Upper path for DIC and
        bleaching fluorophore
     ii. Lower path for observation
        using epi fluorescence

3. Data Analysis

Raw data (5mM taxol on GTP microtubules)

Fluorescence recovery curve:

Amplitude of the gaussians plotted against time

Curve fit to ~e^(-t/t), where t = recovery time

Recovery times compared between:

GMPCPP microtubules with [TAX] from 25pM to 2.5mM
(fill ratio from 0.002 to 0.99)

GTP microtubules with [TAX] from 93nM to 55mM
(fill ratio from 0.03 to 0.94)