Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.
In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteome analysis largely depends on highly sensitive protein detection methods. In this study, a western blot protocol was developed and validated for the detection of beta-actin and the moderately expressed cell death protein caspase-3 in small numbers of cells. Initially, cultured human U937 monocytes and whole sections of paraffin wax-embedded, alcohol-fixed human tonsils were used to optimize protein electrophoresis and western blotting conditions. High-performance NuPAGE Bis-Tris gels in combination with high-quality transfer membranes, optimized antibody concentrations, and a sensitive chemiluminescent substrate provided a strong signal for beta-actin with approximately 500 U937 cells. In the same way, procaspase-3 could be identified with approximately 1000 cells. Similar results were obtained with germinal centre cells that were procured from paraffin wax-embedded, alcohol-fixed human tonsils by LCM. Treatment of U937 cells with etoposide rapidly induced cell death and allowed the detection of active caspase-3 with approximately 2500 cells (0.8 pg of protein). The findings of this study suggest that western blotting is a valuable adjunct to proteome analysis of LCM procured cells.
Division of Pharmacology, University of Antwerp, Wilrijk, Belgium.
Wim
W
Martinet
Vanessa
V
Abbeloos
Nathalie
N
Van Acker
Guido R Y
GR
De Meyer
Arnold G
AG
Herman
Mark M
MM
Kockx
D000199:Actins / Q000032:analysis; D015153:Blotting, Western / Q000379:methods*; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D002452:Cell Count; D002648:Child; D006801:Humans; D042282:Microdissection; D009000:Monocytes / Q000737:chemistry*; D014066:Palatine Tonsil / Q000737:chemistry*; D020298:U937 Cells
D000199:Actins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases