Panga Jaipal Reddy, Aishwarya Anand Rao, Darpan Malhotra, Samridhi Sharma, Ravinder Kumar, Rekha Jain, Kishore Gollapalli, Namita Pendharkar, Srikanth Rapole and Sanjeeva Srivastava Pages 298 - 311 ( 14 )
The success of a proteomic experiment largely depends on the quality and quantity of the protein extract. Currently, various protocols are available for extraction of proteins from different types of samples; however, further optimization is required for every new sample type. Hence, a common protein extraction protocol is desirable. In the present study, soluble proteins were extracted from six diverse samples using TRIzol without any additional clean-up step and subjected to 2-DE and 2D-DIGE analysis for global protein expression profiling. Image analysis using IMP7 and DeCyder showed good coverage, reproducibility and quality of the gel. MS analysis of 24 spots from all the six samples showed good score and coverage for the identified proteins. Additionally, this method facilitated the concurrent isolation of RNA from the same cell lysates with high integrity and quality, suitable for transcriptomic analysis. Thus, we demonstrate the use of a common protein extraction protocol involving TRIzol reagent for 2-DE, 2D-DIGE and MS analysis using six diverse samples and show its suitability for concomitant transcriptomic studies.
2-DE, 2D-DIGE, breast cancer, B. subtilis, glioblastoma cell line, mass spectrometry, protein extraction, S. coelicolor, TRIzol, yeast.
Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400 076, India.