Development of improved cell lysis, solubilization and imaging approaches for proteomic analyses.
Analysis of complex biochemical processes at the level of the proteome requires methods that quantitatively solubilize cytosolic and membrane bound proteins yet are compatible with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, it is often necessary to employ several highly sensitive detection methods to identify key proteins that are modified or exhibit a change in expression levels in response to a given experimental stimulus or condition. Methods were developed that efficiently extract tissues or lyse cultured cells and quantitatively solubilize proteins in a single step without the need to shear nucleic acids. These approaches utilize urea, thiourea, a mixture of detergents, low levels of an ampholyte blend, reductant and a combination of alcohols. To aid in the detection of low abundance proteins and the accurate identification of specific proteins of interest in these samples, two approaches were pursued. In one, proteins are transferred from two-dimensional (2-D) gels to blot membranes. Proteins are then detected by staining with SYPRO Ruby and the resulting 2-D protein pattern is captured using a charge-coupled device (CCD) camera. The blots are then probed with antibodies directed against the protein(s) or functionalities of interest. The resulting chemiluminescent blot image is also generated with the CCD camera and the fluorescent SYPRO Ruby image is recaptured again without moving the membrane. It is thereby possible to generate a direct image overlay of the blot pattern on that of the stained protein pattern. This approach significantly aids in the accurate identification of the dye-stained protein that is detected by the specific antibody. In addition to detecting protein post-gel transfer, a second approach utilizes protein samples labeled with fluorescent dyes prior to 2-D electrophoresis in an effort to increase the sensitivity of protein detection and to facilitate protein quantitation. It is also possible to stain the blots with different dyes and overlay these images as well. Using these approaches, it is possible to perform more rapid and accurate comparative analyses and proteomic, post-gel characterization of proteins of interest than using comparative image analysis of multiple gels.
Leimgruber
Richard M
RM
Pharmacia, Discovery Research and Development, St. Louis, MO 63198, USA. richard.m.leimgruber@pharmacia.com
Malone
James P
JP
Radabaugh
Melissa R
MR
LaPorte
Michelle L
ML
Violand
Bernard N
BN
Monahan
Joseph B
JB
D015153:Blotting, Western; D002232:Carbocyanines; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted; D020543:Proteome / Q000032:analysis*; D012995:Solubility; D020298:U937 Cells
D002232:Carbocyanines; D005456:Fluorescent Dyes; D020543:Proteome; C089187:cyanine dye 3; C085321:cyanine dye 5