Anti-NAB1 | nucleic acid binding protein 1, Chlamydomonas

Samples:
1 - 2 ug of C. reinharditti C1 whole extract
2 - 2 ug of C. reinharditti C1 cell body
3 - 2 ug of C. reinharditti C1 cilia Mark: MW markers 4 - 2 ug of C. reinharditti

Chlamydomonas cilia isolation was performed, and whole cell extract, cell body, and cilia fractions were stored at -80°C. The buffer components consisted of 20 mM HEPES, 10 mM MgCl2, 50 mM KCl, 2 mM DTT, 1 mM EDTA, and 1X Protease inhibitor. Samples were denatured with 6x SDS-Sample Laemmli buffer at 95°C for 5 minutes and store at -20 oC. Samples were separated on 10% SDS-PAGE and transferred overnight to PVDF membranes (pore size of 0.45 μm) using wet transfer in cold conditions. The blot was then blocked with 5% milk for 1 hour at room temperature with agitation. Next, the blot was incubated in the primary antibody at a dilution of 1:1000 for 1 hour at room temperature in TBS-T with agitation. Subsequently, the antibody solution was decanted, and the blot was briefly rinsed twice, followed by three washes for 10 minutes each in TBS-T at room temperature with agitation. The blot was then incubated with goat anti-rabbit IgG secondary antibody (Agrisera goat anti-rabbit IgG horse radish peroxidase conjugated AS09 602) at a dilution of 1:25000 for 1 hour at room temperature with agitation. After incubation with the secondary antibody, the blot was washed three times as previously described and developed using a chemiluminescent detection reagent with exposure time of 30 seconds. 


Courtesy of Meenakshi Tanwar, Department of Cell Biology and Molecular Genetics, University of Maryland, USA