Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane DOI Creative Commons
Takanobu Takata, H Murayama, Togen Masauji

и другие.

BIO-PROTOCOL, Год журнала: 2024, Номер 14(1349)

Опубликована: Янв. 1, 2024

Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) their intermediate/non-enzymatic [e.g., methylglyoxal glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although original required nitrocellulose membranes, we hypothesized that modified contained in may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers unsuitable for this purpose, using an in-house-prepared buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) probes onto PVDF The also entails calculation Tris, CHAPS concentrations, as well protein mass GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used draw a standard curve perform neutralization against non-specific combination anti-GA-AGEs antibodies, thereby enabling quantification GA-AGEs cell lysates. This paper presents detailed protocol analysis GA-AGE levels C2C12 cells. Key features • leverages idea advanced proteins. enables probing Intracellular quantified some types polyclonal anti-GA-AGE antibodies BSA. simultaneously prepared with lysate. There no limit type cultured cells preparation

Язык: Английский

Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane DOI Creative Commons
Takanobu Takata, H Murayama, Togen Masauji

и другие.

BIO-PROTOCOL, Год журнала: 2024, Номер 14(1349)

Опубликована: Янв. 1, 2024

Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) their intermediate/non-enzymatic [e.g., methylglyoxal glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although original required nitrocellulose membranes, we hypothesized that modified contained in may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers unsuitable for this purpose, using an in-house-prepared buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) probes onto PVDF The also entails calculation Tris, CHAPS concentrations, as well protein mass GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used draw a standard curve perform neutralization against non-specific combination anti-GA-AGEs antibodies, thereby enabling quantification GA-AGEs cell lysates. This paper presents detailed protocol analysis GA-AGE levels C2C12 cells. Key features • leverages idea advanced proteins. enables probing Intracellular quantified some types polyclonal anti-GA-AGE antibodies BSA. simultaneously prepared with lysate. There no limit type cultured cells preparation

Язык: Английский

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