Acetylated Tubulin protein Source: Porcine Brain

Acetylated Tubulin protein Source: Porcine Brain

 

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Product Uses Include

  • Acetylated Tubulin Polymerization Studies
  • De-acetylation of Acetylated Tubulin Studies
  • Acetylated Tubulin MAP Binding Studies
  • Mass Spectroscopy of Acetylated Tubulin Studies

Material

The acetylated tubulin protein (Cat. # CS-TAC01) was prepared from porcine brain by adaptation of the method Shelanski et al (6). Further purification was achieved by cation exchange chromatography, and acetylation of K40 of α-tubulin was facilitated by the αTAT1 protein as reported in Friedmann et al and Howes et al (7, 8). Tubulin consists of a heterodimer of one alpha and one beta isotype, each tubulin isotype is 55 kDa in size (see Figure 2). Typically, the molar equivalent of tubulin is defined as the heterodimer which has a molecular weight of 110 kDa. The protein is supplied as a white solid.

Purity
A 10 µg sample of acetylated tubulin protein (Cat. # CS-TAC01) purity is determined by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gradient gel. Acetylated tubulin  protein was determined to be 90% pure.

Picture2_3

Figure 2: A 10 µg sample of acetylated tubulin protein (Cat. # CS-TAC01) purity is determined by scanning densitometry of Coomassie Blue-stained protein on a 4-20% polyacrylamide gradient gel. Acetylated tubulin  protein was determined to be 90% pure. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02). Molecular weight markers are from Invitrogen (Mark 12).

Biological Activity

The biological activity of acetylated tubulin is assessed by a tubulin polymerization assay (Fig. 3). The results indicate K40-Acetylated tubulin shows slightly slower kinetics than native tubulin but it reaches a similar equilibrium point after 900s. 

 

TAC01_biological_activity

Figure 3. Acetylated Tubulin Polymerization Assay 

Each 50 ml well reaction contains 2.5 mg/ml Acetylated tubulin (Cat. # CS-TAC01) (n=2) or 2.5 mg/ml tubulin (Cat. # T240) (n=2). Excitation was at 360 nm and emission at 420 nm measured on an iD5 multi-mode microplate reader (Molecular devices) over 50 minutes at 37oC.

References

  1. Barra H.S., et al. (1974)  Biochem. Biophys. Res. Commun.60:1384-1390.
  2. Eshun-Wilson L., et al. (2019) PNAS 116:10366-10371.
  3. Dompierre et al. (2007) J . Neurosci. 27:3571-3583.
  4. Reed et al. (2006) Curr. Biol. 16:2166-2172.
  5. Nekooki-Machida and Hagiwara. (2020) Med Mol Morphol. 53:191–197.
  6. Shelanski ML, et al. (1973) PNAS 70: 765-768 .
  7. Shida T.., et al. (2010) PNAS 107: 21517-21522
  8. Howes SC., et al. (2014) Mol. Biol. Cell. 25:257-266

 

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