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.

Analysis of Crude, Diverse, and Multiple Advanced Glycation End-Product Patterns May Be Important and Beneficial

“…Kehm et al measured arg-pyrimidine- and pentosidine-positive areas in the pancreatic islets (AGEs area/pancreatic islets) [ 11 ], while van Heijist et al revealed that heat shock protein (HSP) 27 and arg-pyrimidine were co-located in the immunostaining images of squamous cell carcinoma tissue in patients with non-small cell lung cancer; arg-pyrimidine-modified HSP27 was also located in this tissue [ 46 ]. Considering the characteristics that require anti-AGE antibodies, immunostaining [ 2 , 11 , 12 , 46 ], Western blotting [ 2 , 13 , 32 ], slot blotting [ 2 , 14 , 15 , 16 ], and ELISA [ 10 , 17 , 18 ] can be grouped together. Moreover, if the structure of AGEs remains unclear (e.g., AGEs with an unknown structure generated from methylglyoxal and bovine serum albumin [BSA] [ 47 ]), it can still be examined using anti-AGE antibodies produced against the AGEs for the antigen.…”

“…Slot blot analysis is generally performed to quantify AGEs [ 2 , 14 , 15 , 16 ]. In 2017, we developed a novel slot-blot method with the following characteristics: (i) a lysis buffer containing tris-(hydroxymethyl)-aminomethane (Tris), urea, thiourea, and 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and (ii) polyvinylidene fluoride (PVDF) membranes [ 2 ].…”

“…In 2017, we developed a novel slot-blot method with the following characteristics: (i) a lysis buffer containing tris-(hydroxymethyl)-aminomethane (Tris), urea, thiourea, and 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and (ii) polyvinylidene fluoride (PVDF) membranes [ 2 ]. We considered that this lysis buffer could promote the ability of samples containing AGE-modified proteins to suitably probe the PVDF membrane [ 16 ]. We analyzed TAGE and 1,5-anhydro-D-frucotose-derived AGEs (1,5-AF-AGEs) in cells and tissues using TAGE-modified BSA and 1,5-AF-AGEs-modified BSA as standards [ 2 , 14 , 15 , 16 , 50 ].…”

“…We considered that this lysis buffer could promote the ability of samples containing AGE-modified proteins to suitably probe the PVDF membrane [ 16 ]. We analyzed TAGE and 1,5-anhydro-D-frucotose-derived AGEs (1,5-AF-AGEs) in cells and tissues using TAGE-modified BSA and 1,5-AF-AGEs-modified BSA as standards [ 2 , 14 , 15 , 16 , 50 ]. Bronowica-Szyełko et al analyzed serum methylglyoxal-derived AGEs (MGO-AGEs) using slot blotting [ 17 ].…”

“…Although AGEs have been investigated since the early 20th century, when the Maillard reaction was proven [ 9 ], their relationship with human health dates back to ancient times. AGEs have been analyzed using techniques such as fluorimetry [ 10 ], immunostaining [ 2 , 11 , 12 ], Western blotting [ 2 , 13 ], slot blotting [ 2 , 14 , 15 , 16 ], enzyme-linked immunosorbent assay (ELISA) [ 11 , 17 , 18 ], gas chromatography-mass spectrometry (GC-MS) [ 19 ], matrix-assisted laser desorption-mass spectrometry (MALDI-MS) [ 20 ], and electrospray ionization-mass spectrometry (ESI-MS) [ 21 , 22 ]. Researchers have investigated LSRDs and AGEs using these technologies.…”