Koch Lab:Protocols/Kinesin/Tubulin resuspension and polymerization

Steve Koch 00:32, 3 May 2009 (EDT): I am unsure of the historical details of this protocol. It's the one I used in 2003 and 2004, probably adapted from Susan Rivera and Andy Boal. They probably followed protocols from Cytoskeleton or the Kinesin Home Page.

Introduction

This protocol assumes that tubulin has been purchased from Cytoskeleton. Various forms can be purchased. As of 5/2/09, the only two forms we are talking about are lyophilized un-labeled bovine tubulin and rhodamine-labeled bovine tubulin in a 10 mg / ml suspension.

Tubulin dimers are highly unstable, so care should be taken to keep as cold as possible and minimize warm steps except when polymerizing.

Non-labeled tubulin resuspension

1. Tube should contain 1 mg of tubulin.
2. Dissolve the tubulin in 180λ of cold GPEM buffer¹
3. Add 20 λ of cold cushion buffer²
4. Store @ -80C in convenient aliquots. I do 25λ aliquots in 100λ PCR thin-walled tubes.

¹ GPEM is 198λ of BRB80 plus 2λ of 100 mM GTP in 100 mM MgCl2
² I use cushion buffer from cytoskeleton (BST106), I think it is BRB80 w/ 60% glycerol

Rhodamine tubulin resuspension

1. Tube usually contains 2λ of a 10 mg / ml solution of tubulin in GPEM + cushion
2. Spin down several tubes (say 4 tubes) to get liquid to bottom of tube
3. Combine for total volume of say 8 λ.
4. Add 8 λ of GPEM-cushion [9 parts GPEM, 1 part cushion]
5. Store @ -80C in aliquots. I store 1 λ aliquots in 100λ PCR thin-walled tubes.