Visible fluorescent detection of proteins in polyacrylamide gels without staining.
نویسندگان
چکیده
2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.
منابع مشابه
Fluorescent staining for proteins on polyacrylamide gels with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride).
A simple and sensitive post electrophoresis fluorescent staining technique for proteins on polyacrylamide gels using 5-dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) has been developed. Dansyl chloride staining increases the sensitivity, 0.125 micrograms protein per band can be visualised by this technique. The staining method appears to be applicable to all types of proteins i...
متن کاملA Novel Method to Detect β-Cyclodextrin Glucosyl Transferase (β-CGTase) Activity on Polyacrylamide Gels
b-cyclodextrin glucosyl transferase (b-CGTase) hydrolyses starch to produce b-cyclodextrin by transglycosylation (cyclization) activity. The conventional method for detection of b-CGTase activity is based on detecting starch hydrolysis by iodine staining. This method reveals all amylolytic enzymes, but can not discriminate them. In the present study, we introduce a new method for specific detec...
متن کاملImmunofluorescent protein detection in Western blotting
This report describes the detailed procedures for Western blot analysis using fluorescent antibodies. After electrophoresis and subsequent electroblotting, the fluorescent-labeled antibodies were visible upon ultraviolet illumination of the polyvinylidene fluoride (PVDF) membranes and could then be photographed to give an accurate record of the blots. Fluorescent labeling allows for photographi...
متن کاملIdentification of components of protein complexes using a fluorescent photo-cross-linker and mass spectrometry.
This study describes a novel method for improving the specific recognition, detection, and identification of proteins involved in multiprotein complexes. The method is based on a combination of coimmunoprecipitation, chemical cross-linking, and specific fluorescent tagging of protein components in close association with one another. Specific fluorescent tagging of the protein complex components...
متن کاملPerformance characteristics of the High Sensitivity Protein 250 assay for the Agilent 2100 bioanalyzer
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins according to their molecular weights. For highest sensitivity, gels are commonly silver stained using a laborious procedure with low reproducibility, which yields low reproducibility and insufficient quantification capabilities. In this publication we demonstrate the performance of a newly developed Agilent 2100 bioanalyzer me...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Analytical biochemistry
دوره 326 1 شماره
صفحات -
تاریخ انتشار 2004