Biomedical Engineering Reference
In-Depth Information
2.5
2
1.5
1
0.5
0
300
320
340
360
380
400
Wavelength (nm)
Figure 3.7 Fluorescence spectra of human holo- (solid line) and apo- (dashed line)
-lactalbumin recorded
for 20 mM protein solution in 10 mM Tris buffer, pH 8.5, at 20 °C. Fluorescence excitation was at 287 nm
(Barbana et al ., 2008). Reproduced with kind permission from Springer Science and Business Media.
α
holo-
-lactalbumin reaches its maximum at 325 nm. The removal of calcium bound to
the protein causes an increase in fluorescence intensity and shifting of the wavelength
maximum to a significantly higher value (336 nm). These results indicated that tryptophan
residues are more exposed to solvent in the apo form, reflecting a conformational change in
the protein caused by the release of bound calcium (Barbana et al ., 2008 ). Fluorescence
properties of proteins, however, depend on experimental conditions, such as temperature,
pH, concentration, polarity of the environment and quenching (intra- or inter-molecular
interactions) (Andersen et al ., 2008 ).
α
3.5.1.3 Immunochemical methods
Immunochemical methods are highly sensitive techniques for the detection and identification
of protein targets by antigen-antibody specific reactions. Radial immunodiffusion and west-
ern blotting are among the most used techniques for the quantitative estimation of processed
food and bioproduct proteins based on their immunoreactivity with specific antibodies.
Radial immunodiffusion
Radial immunodiffusion is used extensively for the quantitative estimation of antigens. In a
uniformly thin layer of agar containing a specific antibody, the area of the radial diffusion of
the corresponding antigen from wells cut in the gel is directly proportional to the concentra-
tion of the antigen employed. The method is very accurate and sensitive, with lower detection
limits corresponding to an antigen concentration of 1.25 μg/ml (Mancini et al ., 1965 ). This
technique could be used for the determination of antigens and evaluation of the effect of
processing on the denaturation of food proteins (Wehbi et al ., 2005 ).
Western Blotting
After PAGE electrophoretic separation, protein bands can be transferred to an activated
thin support membrane, generally nitrocellulose membrane, by electro-transfer techniques.
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