Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

Abstract

Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry (MS), matrix-associated laser desorption/ionization-MS, and liquid chromatography-electrospray ionization-MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed.

Keywords: 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate; Tris; advanced glycation end products; enzyme-linked immunosorbent assay; gas chromatography–mass spectrometry; liquid chromatography–electrospray ionization–mass spectrometry; polyvinylidene difluoride membrane; slot blot; thiourea; urea.

Figure 1

Bio-Dot SF Microfiltration Apparatus (slot blot apparatus with 48 lanes). (a) Assembly of the apparatus. (b) Parts of the disassembled apparatus.

Figure 2

Setup used for the application of standard TAGE-BSA, HRP marker solution, and sample solution onto the PVDF membrane [27]. White open squares indicate the slot lane. L1, L4: 0, 1, 3, 10, 30, 60, and 100 ng of TAGE-BSA aliquots and HRP marker solution. L2, L3, L5, L6: cell lysate samples of C2C12 cells treated with 0, 0.5, 1, 1.5, and 2 mM glyceraldehyde for 24 h.

Figure 3

Chemiluminescence detection of standard TAGE-BSA aliquots, HRP marker solution, and sample solution on a PVDF membrane [27]. Both membrane sides were simultaneously exposed using Fusion FX. (Left) (L1–L3): Anti-TAGE antibody was probed onto the PVDF membrane. (Right) (L4–L6): Neutralized anti-TAGE antibody was probed onto the PVDF membrane. Closed gray and black squares indicate the bands on the PVDF membrane. L1, L4: 0, 1, 3, 10, 30, 60, and 100 ng of TAGE-BSA aliquots and HRP marker solution. L2, L3, L5, L6: cell lysate samples of C2C12 cells treated with 0, 0.5, 1, 1.5, and 2 mM glyceraldehyde for 24 h.

Figure 4

Model image of western blot with anti-AGE antibody and anti-β-actin. Equal amounts of lysates of hepatic tissues were used. Control: hepatic tissues of rats that had drunk water. HFCS: hepatic tissues of rats that had drunk high-fructose corn syrup (HFCS). Closed gray and black squares indicate the bands on the membrane. The luminance of each band was analyzed and normalized with β-actin. Finally, luminance amounts were quantified.

Figure 5

Model image of the immunostaining of rat hepatic tissue with hematoxylin and eosin, oil red O, and anti-AGE antibody. Control: hepatic tissues of rats that had drunk water. HFCS: hepatic tissues of rats that had drunk high-fructose corn syrup (HFCS). Closed red circles are steatosis drops. Closed brown circles are the areas where anti-AGE antibody was probed.

Figure 6

Model image of preparation of CEL-ester derivative for GC–MS analysis. Closed blue circles are amino acids. Closed peach squares are the compounds which have hydroxyl groups.

Figure 7

Model image of detection of (a) unlabeled CEL-ester derivative (CEL-ester derivative-d₀) in a sample and (b) deuterium CEL-ester derivative (CEL-ester derivative-d₆) as the standard for GC–MS analysis.

Figure 8

Model image of MALDI–MS or ESI–MS analysis of CEL-modified protein.

Figure 9

Hydrochloride (HCl) hydrolysis of various CML-modified proteins and collection of free CML.