Figure 2

Protein blots detection of histone H1 and actin 2 a. Western blot analysis for chemiluminescent detection of histone H1 (around 35 kDa) and actin 2 (around 42 kDa). Nuclear (obtained with the optimized protocol) and cytosolic protein fractions (50 μg) extracted from immature flax seed coats were separated on a 12% SDS-PAGE, transferred to nitrocellulose membrane, and probed with each of the above-noted antibodies. Equal loading of the gels was verified by protein quantization (using fluorescent probe, see Methods) before loading and Coomassie blue staining of the membrane after protein transfer. MW: molecular weight; Ø: Loading buffer without protein (negative control); NPE: nuclear proteins extract (optimized method); Cyt: cytosolic extract (first supernatant, nuclei isolation step). b. Protein Dot blot for chemoluminescent detection of histone H1 was performed using nuclear fraction obtained with the different methods. The color intensity was analyzed and quantified by densitometry using a digital camera and densitometry software. The same letter indicates that values are not significantly different (P > 0.05). Value is mean ± standard error.