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