Preparative denaturing isoelectric focusing for enhancing sensitivity of proteomic studies.
Substantial evidence implicates important roles for both protein phosphorylation and protein degradation in regulation of plant defense responses. Therefore, interest is growing in applying proteomics techniques to investigate these posttranscriptional changes. We have found, however, that most proteins of interest are not visible on two-dimensional (2D) gels without previous prefractionation. This chapter describes the use of preparative denaturing isoelectric focusing to enrich for proteins of specific isoelectric points before separation by 2D gels. This method significantly increases the sensitivity of 2D gel-based comparisons.
Serna-Sanz
Antonio
A
The Sainsbury Laboratory, John Innes Center, Norwich, United Kingdom.
Rairdan
Greg
G
Peck
Scott C
SC
D005591:Chemical Fractionation; D015151:Immunoblotting; D007525:Isoelectric Focusing / Q000379:methods*; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D011489:Protein Denaturation; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity
D010940:Plant Proteins