Assessing the Assembly Competence of Purified Alpha- and Beta-Tubulin Isotypes By: Harjot Kaur University at Buffalo ’12 Mentors: Leah Miller, Berta, Eddie.

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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. –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.

Methodology Purification 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 Marker 1μL2μL4μL5μL

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