Biomedical Engineering Reference
In-Depth Information
Table 4.6
Protein assays
Assays
Applications
References
Western blot
Semi-quantifi cation of protein
expression
Heidebrecht
et al.
, 2009
Flow cytometry
Surface and internal protein
expression assessment
Robins, 1998; Chang
et al.
,
2010
Cell counting
Bodensteiner, 1989
Cell cycle analysis
Pozarowski and
Darzynkiewicz, 2004
Cell sorting
Basu
et al.
, 2010
Colorimetry
Protein concentration
determination
Simonian and Smith, 2006
Protein activity
determination
Gallagher, 2008b
ELISA
Quantifi cation of trace
proteins in solution
Smith, 1993; Sumanas,
2002
4.3.1 Western blot assay
Western blotting or protein immunoblotting is used to identify and semi-
quantify proteins of interest isolated from samples (Heidebrecht
et al.
, 2009).
This assay is especially useful for studying both phosphorylated and non-
phosphorylated proteins isolated from a whole cell or a target sub-cellular
compartment. Western blot assay generally includes four steps: (a) extract
protein and determine concentration, (b) separate protein by gel electro-
phoresis, (c) transfer protein from a gel to a membrane, and (d) identify the
target protein using immuno-detection (Fig. 4.3) (Gallagher, 2008a).
Protein extraction and concentration measurement
Proteins can be isolated from cells or tissues that have been harvested from
two- or three-dimensional culture. Whole cell lysates can be obtained and
prepared by disrupting the cell and nuclear membranes, and extracting sub-
compartmental proteins from the cell membrane, cytoplasm and nucleus. To
prevent protein degradation and maintain phosphorylated state after isola-
tion, proteins should be extracted with proteinase and phosphatase inhibi-
tors and kept on ice during the preparation.
Varieties of commercial protein extraction kits are available for total or
subcompartmental protein isolation, which can signifi cantly increase extrac-
tion effi ciency and improve sample quality (Millipore, 2010; QIAGEN,
2010d; ThermoFisherScientifi c, 2010a, 2010b). Before electrophoresis, the
protein concentration should be quantifi ed for the equal loading of pro-
tein for each sample. Colorimetric methods, such as the BCA, BradFord or
