Chemiluminescent standards for quantitative comparison of two-dimensional electrophoresis western blots.
Chemiluminescent probes offer highly sensitive quantitative analyses of proteins blotted from electrophoretic gels onto a supporting matrix (e.g. nitrocellulose or polyvinylidene difluoride). Visualization of signals from probes involves the emission of light that is dependent on a number of variables (e.g. conjugated enzyme activity, antibody titer, hybridization efficiency, substrate concentration, chemical reactivity, temperature, etc.). Thus, it is important to be able to correct for these variations. For example, the exposure time of the blot to the detection medium (e.g., film or digital camera) is a critical variable in the final results. Several protein samples separated on a single blot (e.g. one-dimensional resolution) can be compared from the ratio of the individual proteins, but comparison of separate blots completed on different days requires a chemiluminescent standard. The situation is more complex when only one sample per gel/blot is used (i.e. two-dimensional electrophoresis (2-DE)). This paper describes a method for preparing agarose embedded standardized strips that contain dilutions of antigens that can be visualized with the corresponding probe antibody. This standardization strip can be produced from common laboratory supplies and provides a method to correct for alterations in chemiluminescent intensities from different 2-DE analysis. Several standardization strips can be produced simultaneously and then used during the electroblotting step of different blots on different days.
Conrad
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CC
Molecular Aging Unit, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA.
Malakowsky
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CA
Talent
J
J
Rong
D
D
Lakdawala
S
S
Gracy
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RW
D000818:Animals; D000906:Antibodies; D000941:Antigens / Q000302:isolation & purification; D015153:Blotting, Western / Q000592:standards*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000592:standards*; D006801:Humans; D008163:Luminescent Measurements; D020543:Proteome; D012015:Reference Standards
D000906:Antibodies; D000941:Antigens; D020543:Proteome