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, adapted from George Bachand and probably from Susan Rivera and Andy Boal. They probably followed protocols from Cytoskeleton or the Kinesin Home Page, probably a Howard Lab protocol?

Introduction

This protocol assumes that tubulin has been purchased from Cytoskeleton. While various forms of tubulin can be purchased, we use lyophilized un-labeled bovine tubulin and lyophilized rhodamine-labeled bovine tubulin.

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

Materials

Tubulin

Tubulin can be purified in-house from various sources, typically bovine brain. Steve's experience is with using commercial tubulin from Cytoskeleton. Significant hurdles still remain for recombinant tubulin production.

Right now, we use the following tubulin products from Cytoskeleton.

• non-labeled lyophilized tubulin
• Rhodamine tubulin products

Buffers

The naming convention for our buffers is not standard. However, for convenience and to facilitate lab communication, we use the following naming scheme. All buffers have PIPES, EGTA, and MgCl₂ in them and various additions to the base buffer. The additions are followed by the PEM label with an abbreviation to the chemical name.

• PEM
• PEM-G
• PEM-A
• PEM-Gly60
• PEM-Gly6

Un-labeled tubulin resuspension

Un-labeled tubulin comes packed in vials containing 1 mg of tubulin. We resuspend this tubulin to a final concentration of 5 mg/mL.

Required buffers

• PEM-G
• PEM-Gly60.

Procedure

1. Remove a vial of tubulin from the -80˚C freezer and place on ice or put in the e•IceBucket and defrost.
   1. If necessary, spin the vial to get all the tubulin to settle at the bottom. Be careful though since tubulin is very labile and may be destroyed during this step.
2. Make sure you have enough PEM-G and PEM-Gly60 for resuspension. Place these buffers on ice or in the e•IceBucket as well.
3. Resuspend the tubulin in 180 µL of cold PEM-G.
4. Add 20 µL of cold PEM-Gly60.
5. Store in the -80˚C freezer in convenient aliquots.

Rhodamine tubulin resuspension

Rhodamine labeled tubulin comes packed in vials containing 20 µg of tubulin. We resuspend this tubulin to a final concentration of 5 mg/mL.

Required buffers

• GTP.
• PEM.
• PEM-Gly60.

Procedure

1. Remove a vial of tubulin from the -80˚C freezer and place on ice or put in the e•IceBucket and defrost.
   1. If necessary, spin the vial to get all the tubulin to settle at the bottom. Be careful though since tubulin is very labile and may be destroyed during this step.
2. Prepare a solution of PEM-Gly6 with 1 mM GTP in it and store on ice or in the e•IceBucket.
   1. 1 µL of GTP
   2. 10 µL of PEM-Gly60
   3. 89 µL of PEM
3. Resuspend the tubulin in 4 µL of cold PEM-Gly6 with 1 mM GTP.
4. Store in the -80˚C freezer in convenient aliquots.

Microtubule polymerization

1. Because I store my aliquots in PCR thin-walled tubes, I can pop them right into the thermal cycler w/ hot lid. The hot-lid prevents very small volumes from drying out during polymerization.
   • Steve Koch 00:51, 3 May 2009 (EDT): Note: I believe we often did not mix unlabeled with rhodamine-labeled tubulin, and often used 100% rhodamine-labeled tubulin. (Or, rather 100% coming from that cytoskeleton product, probably not 100% efficient, though.) It's probably a much better idea to mix rhodamine-labeled tubulin with un-labeled before polymerizing. At the moment, I don't have the proper ratios for this procedure.
2. Pre-heat thermal cycler to 37°C w/ hot-lid ON.
3. Heat tubes at 37°C for 20 minutes.
4. Add whatever volume of BRB80T (BRB80 buffer with Taxol) appropriate for further experiments. For unlabeled tubulin, I usually add 75 λ of BRB80T to 25 λ of polymerized MTs. For rhodamine tubulin, I think we usually add 100λ of BRB80T. One can use 37°C BRB80T to be extra-careful, but I usually use room temp.
5. Procede with experiments, or biospin clean-up

Optional Bio-Spin Column

1. The Bio-rad Bio-spin 6 columns can be used to get rid of contaminating Pi (inorganic phosphate) or rhodamine dye, or to shear the MTs
   • Steve Koch 00:59, 3 May 2009 (EDT): I never confirmed the ability of the spin column to shear the MTs and I was highly skeptical. Getting rid of Pi is useful for malachite green kinesin activity assays.
2. I re-use the bio-spin 6 column, effectively using a column already blocked with tubulin.
3. For first preparation of column, I will first block with 200 λ of 2mg / ml (or higher) BSA. Then 500 λ BRB80T, then 100λ of MTs (10 μM), then 4 times 500 λ BRB80T. For an already-used column, I will just do 4 x 500λ BRB80T to equilibrate. Spins are usually in the small fixed-speed table-top centrifuge (Galaxy D?) 30 – 60 seconds.
4. Load the MTs (<100λ) onto the column, spin for 60 seconds to recover MTs.

Other references

This page was initially prepared based on my Word document notes from Sandia. Ultimately I'd love to have all of them converted to OWW. Until then, I'm manually uploading a few to GoogleDocs. Here's two used to prepare this page:

• http://docs.google.com/Doc?id=dgqjkh6p_83dz57zchd
• http://docs.google.com/Doc?id=dgqjkh6p_84fbwgcgf8

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