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
The protocols below assume 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 them as cold as possible and to minimize warm steps, except when polymerizing.

Materials
The materials used for tubulin resuspension can be purchased completely from Cytoskeleton. We make our own buffers and thus do not purchase everything from them. Those items not purchased from Cytoskeleton are noted as such.

Tubulin
Tubulin can be purified in-house from various sources, typically bovine brain. Steve's experience is with using commercial tubulin from Cytoskeleton and thus we do not purify our own. While it is possible to recombinantly grow tubulin, significant hurdles still remain for recombinant tubulin production.

We use the following tubulin products from Cytoskeleton.


 * Non-labeled lyophilized bovine tubulin
 * Rhodamine-labeled bovine tubulin

Buffers
The naming convention for our buffers is not standard. You will see in the literature what we call PEM, named as BRB80. Please see here for a discussion of the buffer naming convention used in the literature. For convenience, and to facilitate lab communication, we use the following naming scheme. All buffers consist of a base of chemicals. Those chemicals are PIPES, EGTA, and MgCl2. Any additions to the base PEM construct are labeled by following the PEM label with an abbreviation of the added chemical name. The buffers we use include,


 * PEM
 * PEM-G
 * PEM-Gly60
 * PEM-T

The required chemicals for the above buffers were either purchased from Sigma-Aldrich or Cytoskeleton and they include, It should be noted that Cytoskeleton sells what we call PEM-Gly60.
 * |SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC PIPES
 * |FLUKA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC EGTA
 * |SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC MgCl2
 * GTP
 * Taxol - Comes with DMSO for suspension of Taxol.
 * |SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Glycerol

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.
 * 2) 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.
 * 3) Make sure you have enough PEM-G and PEM-Gly60 for resuspension. Place these buffers on ice or in the e•IceBucket as well.
 * 4) Resuspend the tubulin in 180 µL of cold PEM-G.
 * 5) Add 20 µL of cold PEM-Gly60.
 * 6) Store in the -80˚C freezer in convenient aliquots. We typically make 5 µL aliquots.

Final solution

The final solution should contain:
 * 80 mM PIPES
 * 1 mM EGTA
 * 1 mM MgCl2
 * 1 mM GTP
 * 6% (v/v) Glycerol
 * 5 mg/mL un-labeled tubulin

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


 * PEM-G
 * PEM-Gly60

Procedure

You can also prepare partially labeled tubulin if you have un-labeled tubulin prepared ahead of time. Since we prepare un-labeled tubulin in 5 µL aliquots, adding a 1 µL aliquot of rhodamine-labeled tubulin to the un-labeled tubulin will give you a sample with tubulin that is approximately 17% labeled with rhodamine. We then take our partially labeled tubulin prep and aliquot it into 1 µL samples for later polymerization.
 * 1) Remove a vial of tubulin from the -80˚C freezer and place on ice or put in the e•IceBucket and defrost.
 * 2) 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.
 * 3) Prepare a solution of PEM-Gly6 with 1 mM GTP in it and store on ice or in the e•IceBucket.
 * 4) 1 µL of GTP
 * 5) 10 µL of PEM-Gly60
 * 6) 89 µL of PEM
 * 7) Resuspend the tubulin in 4 µL of cold PEM-Gly6 with 1 mM GTP.
 * 8) Store in the -80˚C freezer in convenient aliquots. We typically make 1 µL aliquots.

Final solution

The final solution should contain:
 * 80 mM PIPES
 * 1 mM EGTA
 * 1 mM MgCl2
 * 1 mM GTP
 * 6% (v/v) Glycerol
 * 5 mg/mL rhodamine-labeled tubulin

Microtubule polymerization
Removal of tubulin from the -80˚C freezer should only be done if it is to be polymerized immediately.

Required buffers


 * PEM-T

Procedure


 * 1) Pre-heat the thermal cycler to 37°C. Ensure that the hot-lid is ON. The hot-lid prevents very small volumes from drying out during polymerization and is essential to ensure polymerization.
 * 2) Keep in the thermal cycler for 20 minutes.
 * 3) Add whatever volume of PEM-T appropriate for further experiments. One can use 37°C PEM-T to be extra-careful, however, room temperature PEM-T is fine.
 * 4) For un-labeled tubulin, add 75 µL of PEM-T to 25 µL of polymerized microtubules.
 * 5) For rhodamine-labeled tubulin, add 199 µL of PEM-T.
 * 6) Proceed with experiments, or use the Bio-spin clean-up procedure explained below.

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
 * 2) * 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.
 * 3) I re-use the bio-spin 6 column, effectively using a column already blocked with tubulin.
 * 4) 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.
 * 5) Load the MTs (<100λ) onto the column, spin for 60 seconds to recover MTs.

Other references
This page was initially prepared based on Koch's 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