Anti-Tubulin alpha chain (monoclonal antibody)

Samples:

1 - 14 µg of Nicotiana tabacum, L. whole leaf extract of adult control plant (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 Chlorella vulgaris extract- control (untreated)
9 – 7 µg of Arabidopsis thaliana seedlings extract- control (untreated)

14 µg/well of total protein extracted freshly from 0.05 g of lyophilized samples as specified above, following the Phenol extraction protocol 1 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 1:8000 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.

Metal induced stress affected the expression of tubulin, and that therefore, this protein cannot be used as a loading control under that type of conditions

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