TY  - JOUR
TI  - Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane
AU  - Takata, Takanobu 
AU  - Murayama, Hiroki 
AU  - Masauji, Togen 
VL  - 14
IS  - 14
PY  - 2024
DA  - 2024/07/20
SP  - e5038
C1  - Bio-protocol 2024;14:e5038
DO  - 10.21769/BioProtoc.5038
UR  - https://doi.org/10.21769/BioProtoc.5038
AB  - Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells.
KW  - Advanced glycation end product
KW  - Glyceraldehyde
KW  - GA-AGE
KW  - Polyvinylidene difluoride
KW  - 2-amino-2-hydromethyl-1
KW  - 3-propanediol
KW  - Urea
KW  - Thiourea
KW  - 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate
KW  - Slot blot analysis
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False