Assessing the Assembly Competence of Purified Alpha-

and Beta-Tubulin Isotypes

By: Harjot KaurUniversity at Buffalo ’12

Mentors: Leah Miller, Berta, Eddie NievesAlbert Einstein College of Medicine

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

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.

Purpose

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

MethodologyPurification of Tubulin from

Chicken Erythrocytes

Run the extract in a SDS gel and through FPLC

Perfrom Western Blotting and run the samples through Mass spectrometer

Run the sample through Phophocellulose column

Data Analysis

Different Concentrations of Tubulin

Mar

ker

1μL 2μL 4μL 5μL

Phosphocellulose Column GelC

ontro

l1 2 3 4 5 6 Pe

llet

1= Extract 12= Extract 23= Extract 34= Extract 45= Extract 56= Extract 6

Western Blotting Alpha Antibody

Western Blotting Beta Antibody

Results for Control Maldi TOF/TOF

Results for Empty Maldi TOF/TOF

Results for A12 Maldi TOF/TOF

Results for B12 Maldi TOF/TOF

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.

• 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

Acknowledgements