TY - JOUR
T1 - An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis
AU - Wang, Sheng Bing
AU - Hu, Qiang
AU - Sommerfeld, Milton
AU - Chen, Feng
PY - 2003/11/1
Y1 - 2003/11/1
N2 - Two-dimensional gel electrophoresis (2-DE) is a core proteomic technique to study protein expression and function in living organisms. Although it has been extensively used for investigation of bacterial, yeast, animal and plant tissue cells, there is limited information about the use of 2-DE in microalgal research. In this study, a number of key chemical reagents, including acetone, trichloroacetic acid, urea, thiourea, dithiothreitol, and tributyl phosphine, were quantitatively evaluated for 2-DE of green microalgae, using Haematococcus pluvialis as a model system. The goal was to maximize the number and staining intensity of protein spots while minimizing streaking and smearing on the second dimensional SDS gel. Compared to non-frozen immobilized pH gradients (IPG) strips, freezing of the IPG strips at -20°C after isoelectric focusing (IEF) enhanced protein resolubilization and transfer into the SDS gel, and thus improved resolution while eliminating vertical point streaking on the SDS gel. It was also confirmed that manipulation of sample loading capacity is a simple, effective purification strategy for selective investigation of the proteins of interest and of varying abundances. The protocol was also successfully applied to profiling protein expression in H. pluvialis under external stress conditions, indicating its potential usefulness in further proteomics studies of this organism and related species.
AB - Two-dimensional gel electrophoresis (2-DE) is a core proteomic technique to study protein expression and function in living organisms. Although it has been extensively used for investigation of bacterial, yeast, animal and plant tissue cells, there is limited information about the use of 2-DE in microalgal research. In this study, a number of key chemical reagents, including acetone, trichloroacetic acid, urea, thiourea, dithiothreitol, and tributyl phosphine, were quantitatively evaluated for 2-DE of green microalgae, using Haematococcus pluvialis as a model system. The goal was to maximize the number and staining intensity of protein spots while minimizing streaking and smearing on the second dimensional SDS gel. Compared to non-frozen immobilized pH gradients (IPG) strips, freezing of the IPG strips at -20°C after isoelectric focusing (IEF) enhanced protein resolubilization and transfer into the SDS gel, and thus improved resolution while eliminating vertical point streaking on the SDS gel. It was also confirmed that manipulation of sample loading capacity is a simple, effective purification strategy for selective investigation of the proteins of interest and of varying abundances. The protocol was also successfully applied to profiling protein expression in H. pluvialis under external stress conditions, indicating its potential usefulness in further proteomics studies of this organism and related species.
KW - Green algae
KW - Haematococcus pluvialis
KW - Proteomics
KW - Thiourea
KW - Tributyl phosphine
KW - Two-dimensional gel electrophoresis
UR - http://www.scopus.com/inward/record.url?scp=0348011459&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0348011459&partnerID=8YFLogxK
U2 - 10.1023/B:JAPH.0000004324.88897.b2
DO - 10.1023/B:JAPH.0000004324.88897.b2
M3 - Article
AN - SCOPUS:0348011459
SN - 0921-8971
VL - 15
SP - 485
EP - 496
JO - Journal of Applied Phycology
JF - Journal of Applied Phycology
IS - 6
ER -