A novel probe for chemiluminescent image detection of proteins in two‐dimensional gel electrophoresis

Abstract

Carrier ampholytes were found to enhance the chemiluminescence (CL) emission from the 3-aminophthalic hydrazide (luminol)-hydrogen peroxide system. They can be used as a chemiluminescent probe for rapid detection of major proteins in gels. This probe attracted much interest due to its ability to attach proteins, and to the possibility to combine it with separation techniques generating the CL emission directly. Increased signal intensity was achieved employing optimized concentrations of the carrier ampholyte enhancer. The binding of carrier ampholyte to proteins was found to occur at the pI of the proteins. Proteins from different regions of the gels were identified by their matrix-assisted TOF mass spectra and by appropriate database search, the results illustrating the possibility of major protein detection in human serum. Direct CL image detection with the carrier ampholyte probe can be applied for the detection of characteristic proteins in patients, i.e., proteins which cannot be detected without the probe.

Development of sensitive metalloporphyrin probes for chemiluminescent imaging detection of serum proteins

“…Furthermore, metalloporphyrins can be one‐step labeled to proteins without the requirement of special instruments. 12, 13…”

“…Nondenaturing 1‐DE and 2‐DE were carried out as previously described 12. Briefly, the 1‐DE was performed at a separation temperature of 20°C in a vertical discontinuous gel system, consisting of separating (7.5%, m / v ) and stacking (4.0%, m / v ) gels (82 mm×82 mm×1.5 mm).…”

“…Unfortunately, most proteins cannot generate CL emission, and this disadvantage confined the method to clinical applications to some extent. Hence, much work has been done to develop a more sensitive and comprehensive CL imaging detection method 10–13.…”

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis

“…Nondenaturing 1D and 2D polyacrylamide gel electrophoresis was carried out as previously described. 20 For 1D-PAGE, serum (40 mL) was mixed with 360 mL of loading buffer, which was composed of 80 mL bromophenol blue (0.02%, w/v), 80 mL glycerol (20%, v/v), and 200 mL H 2 O; the loading volume for each well was 15 mL. The voltage as the samples migrated through the stacking gels was 120 V. When the samples entered the resolving gels, the voltage was decreased to 100 V and was kept constant for 2.5 h. For 2D-PAGE, 15 mL human serum was diluted with the loading buffer (glycerol, bromophenol blue and deionized water) to 90 mL.…”

Novel Application of Carbon Nanotubes for Improving Resolution in Detecting Human Serum Proteins with Native Polyacrylamide Gel Electrophoresis

“…Complement C3 is mostly synthesized in the liver and correlates with features of the insulin resistance syndrome and predicts the development of diabetes. It is of great diagnostic importance and may indicate progression of atherosclerosis and act as a specific marker of chronic inflammation. , Apo A-I and Apo A-IV are prominent protein constituents of high-density lipoproteins (HDL) and are involved in reverse cholesterol transport to a different extent. They are potent cofactors for the lecithin- cholesterol acyltransferase reaction and bind to cholesterol-loaded cells to promote cellular cholesterol efflux.…”