The importance of choice of visualization technique in the use of indirect immunodetection methods: specific reference to the detection of light chain movement on a regulatory myosin.
This paper addresses a practical problem associated with the use of visual detection systems used in immunoblotting. Western blot analyses from the same experiment, differing only at the level of the secondary antibody used and the means of visualization employed, have produced apparently different results which, in isolation, could lead to different conclusions at both the qualitative and quantitative level. Indirect immunofluorescence, using a fluorescein isothiocyanate labeled secondary antibody and visualized by fluorescence excitation, was excellent for detecting the major species present but could not detect minor components. Indirect immunoperoxidase staining, on the other hand, appeared to detect all immunoreactive species present, both major and minor, presumably reflecting a more realistic picture of the experimental situation. All results were obtained during the observation of photocross-link formation between regulatory light chains and regulatory and essential light chains after hybridization of scallop myosin with benzophenone-4-maleimide labeled regulatory light chains. Results were obtained under conditions designed to simulate the physiological states of rest and rigor; the implication of these results with respect to myosin-linked regulation is discussed.
Bower
S M
SM
Department of Anatomy, Medical College of Pennsylvania, Philadelphia 19129.
Chantler
P D
PD
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003445:Crustacea; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques; D008856:Microscopy, Fluorescence; D009210:Myofibrils / Q000737:chemistry; D009218:Myosins / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D009218:Myosins