Tubulin gamma chain (monoclonal antibodies)

Samples:
1 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant- control (untreated)
2 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant after treatment with 100 µM AgNP-PVP
3 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant after treatment with 100 µM AgNP-CTAB
4 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant after treatment with 100 µM AgNO3
5 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant after treatment with 100 µM AgNP-PVP+cys
6 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant after treatment with 100 µM AgNP-CTAB+cys
7 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult plant after treatment with 100 µM AgNO3+cys
8 - 14 µg of Nicotiana tabacum, L. extract of 4 weeks old seedlings- control (untreated)
9 – 14 µg of Chlorella vulgaris extract- control (untreated)

14 µg/well of total protein extracted freshly from 0.05 g of lyophilized whole leaf and root of adult tobacco plants following the Phenol extraction protocol1 with extraction buffer containing Trizma base (500 mM), Ethylenediaminetetraacetic acid (EDTA) (50 mM), sucrose (700 mM) and Potassium chloride (KCl) (100 Mm) with addition of phenylmethylsulfonyl fluoride (PMSF) (1mM) and 2% β-mercaptoethanol. After short incubation on 4°C with agitation, phenol was added. The phenol (supernatant) phase containing proteins, was collected after centrifugation and equal volume of extraction buffer was added. After centrifugation, supernatant phase was collected and 4 volumes of 0.1 M ammonium acetate (with 10% methanol) was added, and proteins were precipitated ON/-20°C. The next day, protein pellets were washed 3 times in ammonium acetate with rounds of centrifugations in between, and finally in aceton. Protein pellet was lastly resuspended in Isoelectric focusing buffer (IEF) containing 9 M urea, 4% CHAPS, 20 mM DTT, 1.2% Ampholytes pH 3 to 10. Protein concentrations was measured with modified Bradford method1 and denatured with Laemmli sample buffer2 at 95°C for 5 min. Total proteins were separated on 12 % SDS-PAGE and blotted 1h to nitrocellulose (pore size of 0.2 µm), using wet transfer. Blot was blocked with 2 % milk in PBS-T, 1h/RT. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h/RT with agitation in a solution of 2 % milk in PBS-T and then ON/4°C. The antibody solution was decanted and the blot was then washed 3 times for 10 min in 2% milk in PBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (goat anti-Mouse IgG1 HRP conjugated, AS16 3715) diluted to 1:10 000 in 2% milk in PBS-T for 1h/RT with agitation. The blot was washed twice for 10 min in PBS-T developed for 5 min with AgriseraECLSuperBright (AS16 ECL-S). Exposure time was 12 min.

Courtesty of MSc, Karla Košpić, University of Zagreb, Faculty of Science Department of Biology, Croatia

Tubulin gamma chain is essential for the control of microtubular network. It is found at microtubule organizing centers (MTOC) such as the spindle poles