Biomaterial Lab Protocols

Here is a collection of lab protocols that I have worked to develop for experiments in the Dogic Lab and Fredberg Lab. This page is a resource for members of the research community who may be interested in experiments in biomaterial science dealing with surface coatings and functionalization, protein purification, and cell culture. 

Acrylamide Coated Glass
Passivation with a steric Polymer Brush.

We use this protocol is apply a coating of a sterically repulsive, non-interacting acrylamide polymer brush onto microscope glass in order to passivate the glass against protein adhesion.

PEG Coated Glass

We use this protocol is apply a coating of a sterically repulsive, non-interacting PEG polymer brush onto microscope glass in order to passivate the glass against protein adhesion.

Flat Oil-Water Interfaces

Use this protocol to create a 2D large-area but micron-thin layer of (fluorinated) oil in a ~100um thick microscope chamber. This surface is perfect for performing experiments at surfactant stabilized liquid-liquid interfaces while the surfactant ensures that the surface/interface remains biocompatible. We use it for a 2D active nematic liquid crystal composed of cytoskeletal proteins.

Passive Tracer Particles

Passivate micron sized tracer particles (microspheres) with a functionalized poly-Lysine group followed by a passive PEG brush. The coating works on 3um and 200nm microspheres and uses economical and commercially available reagents.  The spheres have been used in microtubule based experiments in Dogic Lab and have been found to not interact (stick) with the protein filaments.

Tubulin Protein From Brain Purification

This protocol is adapted from the 2003 Popov Tubulin Purification protocol which purifies tubulin using a high molarity PIPES buffer. Here, I have adapted this protocol for Dogic Lab into step-by-step directions along with notes at each step from one of the early preps.

Tubulin Protein Recycling Guide

Once tubulin protein is purified, it should be recycled so that only polymerization-competent tubulin remains in the final stock before use in experiments. This protocol is adapted from the widely used Tim Mitchison Lab tubulin and microtubule protocols as a step-by-step guide for the Dogic Lab.

Tubulin Protein Labeling Guide

To use tubulin or microtubules in fluorescence microscopy experiments, I use this protocol to label the tubulin with Alexa 647 dye. The protocol is adapted from the widely used Tim Mitchison Lab tubulin and microtubule protocols as a step-by-step buide for labeling tubulin in the Dogic Lab. This protocol is for a large amount of tubulin and requires a significant amount of dye. It can be scaled to smaller preps easily.

MDCK Cell Media Recipe

MDCK Cell Passage Protocol

Glass - Acrylamide Gel Coating
for epithelial cell culture

Acrylamide Gel Functionalization
Collagen I
for epithelial cell culture

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Stephen J. DeCamp