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Assessing the Assembly Competence of Purified Alpha- and Beta- Tubulin Isotypes

Assessing the Assembly Competence of Purified Alpha- and Beta- Tubulin Isotypes. By: Harjot Kaur University at Buffalo ’12 Mentors: Leah Miller, Berta, Eddie Nieves Albert Einstein College of Medicine. Background. Microtubules consist of two protein subunits: α-tubulin and β-tubulin.

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Assessing the Assembly Competence of Purified Alpha- and Beta- Tubulin Isotypes

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  1. Assessing the Assembly Competence of Purified Alpha- and Beta-TubulinIsotypes By: Harjot Kaur University at Buffalo ’12 Mentors: Leah Miller, Berta, Eddie Nieves Albert Einstein College of Medicine

  2. Background • Microtubules consist of two protein subunits: α-tubulin and β-tubulin. • In mammals there are 6 α- and 7 β-tubulin protein forms that differ mainly at the C-terminus • These different protein forms are called isotypes

  3. Microtubules This hollow bead like structure is alpha and beta tubulin coming together to form microtubules. Due to the alternation between alpha and beta tubulin, it is highly likeable for one end of the microtubules to be alpha and the opposite end to be beta.

  4. Purpose • Separating α- and β-tubulin from microtubules and being able to reassemble them into the same bead-like structure.

  5. Run the sample through Phophocellulose column Run the extract in a SDS gel and through FPLC Data Analysis Methodology Purification of Tubulin from Chicken Erythrocytes Perfrom Western Blotting and run the samples through Mass spectrometer

  6. Different Concentrations of Tubulin Marker 1μL 2μL 4μL 5μL

  7. Phosphocellulose Column Gel Control Pellet 1 2 3 4 5 6 1= Extract 1 2= Extract 2 3= Extract 3 4= Extract 4 5= Extract 5 6= Extract 6

  8. Western Blotting Alpha Antibody

  9. Western Blotting Beta Antibody

  10. Results for Control Maldi TOF/TOF

  11. Results for Empty Maldi TOF/TOF

  12. Results for A12 Maldi TOF/TOF

  13. Results for B12 Maldi TOF/TOF

  14. Future Studies Currently, we are re-doing the western blotting and the mass spec. due to the contamination of alpha in the beta and vice versa. We also hope to overcome the difficulties of reassembling the alpha and beta-tubulin into microtubules.

  15. Acknowledgements • Leah Miller • Berta Burd • Eddie Nieves • Dr. Ruth Angeletti • Everyone in the lab • Dr. Sat Bhattacharya • Albert Einstein College of Medicine • Harlem Children Society and Staff

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