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Large Scale Tubulin Preparation

2019.4.21

Tubulin is purified from bovine/porcine brain by two cycles of polymerization/depolymerization followed by removal of copurifying proteins on a phosphocellulose (PC) column. The procedure described here is for a large scale prep (10 cow brains) that yields 1-4 grams of tubulin. The protocol can be scaled down if such a large prep is either not necessary or not doable. For ease of organization, all the pre-prep and day-of-prep activities are listed in outline form before details about the prep itself.

Back to protocols

I. Tubulin Prep Outline

Pre-Prep:

1. Call slaughterhouse and request fresh brains to be picked up the morning of prep

2. Pour and equilibrate phosphocellulose column

3. Make buffers

4. Ensure that reagents, such as ATP and GTP, are present in sufficient

amount for the prep

5. Sign up for centrifuges and rotors and gather centrifuge tubes,

blenders and motorized homogenizer/dounce

6. Prepare cooler for transporting brains night before prep and organize

coldroom for morning mayhem

Prep Day:

7. Remove meninges, brain stems and blood clots, weigh and

homogenize brains in blenders

8. Clarify homogenate and use supernatant for 1st polymerization cycle

9. Collect 1st cycle polymer fraction by centrifugation

10. Depolymerize 1st cycle polymer by homogenization at 0-4⁰C

11. Clarify depolymerization mix and use supernatant for 2nd

polymerization cycle

12. Collect 2nd cycle polymer fraction by centrifugation

13. Depolymerize 2nd cycle polymer by homogenization at 0-4⁰C

14. Clarify depolymerization mix and load supernatant onto PC

column

15. Collect flowthrough from PC column, aliquot and freeze at -80⁰C

II. Buffers & Nucleotides

PB (Pipes/Polymerization Buffer): 0.1 M K-Pipes, pH 6.8, 0.5 mM MgCl₂, 2 mM EGTA, 0.1 mM EDTA, 0.1 % b-mercaptoethanol, 1 mM ATP. Need 8 liters in coldroom
(Add ATP and BME just prior to beginning the prep)

CB (Column Buffer): 50 mM K-Pipes, pH 6.8; 1 mM EGTA; 0.2 mM MgCl₂. Need ~25 liters for equilibration, running and storage of PC column

CB + 1 M KCl: Need ~10 liters for prewashing and eluting the PC column

To make 1L of 10X CB:

151.2 grams PIPES, free acid
3.8 grams EGTA
2 ml of 1 M MgCl₂
pH with KOH to pH 6.75, and bring up to 1 liter.

Check pH at 1X is 6.7 Make 3.5 liters of 10X CB for 10-12 brain prep.

GTP: Sigma Type IIS- # G-8752

ATP: Sigma Grade 1- # A-2383

Glycerol: 2-3L prewarmed to 37¡C (store overnight in 37¡C incubator)