oxiselecttm advanced glycation end product competitive elisa kit  (Cell Biolabs Inc)

Journal: Scientific Reports

Article Title: Glycation damage to organelles and their DNA increases during maize seedling development

doi: 10.1038/s41598-022-06454-7

Figure Lengend Snippet: Key steps in glycation process. Reducing sugars interact with amino groups of protein or nucleic acids to form unstable Schiff bases that undergo intramolecular rearrangement to form Amadori products (early glycation products) such as fructosamine. These products are converted to reactive dicarbonyl compounds (intermediate glycation products) such as methylglyoxal (MGO), glyoxal, and 3-deoxyglucosone that are then irreversibly converted into advanced glycation products (AGEs). All the reactions are spontaneous (not enzymatically catalyzed). Further details are provided in refs 10, 14, and 46. Examples of AGE adducts to proteins and DNA: CML: Nε-(carboxymethyl)-lysine; CEL: Nε-(carboxyethyl)-L-lysine; CEdG: N2-(1-carboxyethyl)-deoxyguanosine; dG-MG: 3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one.

Article Snippet: AGE in plastid and mitochondrial proteins was determined using the OxiSelectTM Advanced Glycation End Product (AGE) Competitive ELISA Kit (Cell Biolabs Inc.; cat # STA-817) as per manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Glycation damage to organelles and their DNA increases during maize seedling development

doi: 10.1038/s41598-022-06454-7

Figure Lengend Snippet: AGE in maize mitochondria and plastids. The amount of AGE in proteins extracted from isolated mitochondria and plastids was measured using anti-AGE antibodies with a competitive ELISA ( A – D ) and slot blot ( E – L ) assays. For the ELISA assay the levels of AGE in total mitochondrial and plastid proteins were measured as µg/mL. Representative images of slot blot assays are shown in ( I–L ) and the relative AGE in protein was determined, where the image intensity was set to 1 for stalk lower in ( E , G ) and for dark-grown leaves in ( F , H ). Uppermost panels of ( I – L ) show AGE proteins, middle panels show the total protein (TP) levels in samples, and lowermost panels show mitochondrial (COX2)- and plastid (RbcL)-specific proteins (COX2 in panels I and J; RbcL in panels K and L). Equal amounts of protein extracted from the different tissues were used in these assays. Data here and in Figs. , , , are represented as mean ± SEM. Statistically significant differences were measured using ANOVA statistic test with post hoc analysis using Tukey’s HSD and are shown as asterisks, where * P -value ≤ 0.05, ** P -value ≤ 0.01, *** P -value ≤ 0.001 are indicated on respective graphs.

Article Snippet: AGE in plastid and mitochondrial proteins was determined using the OxiSelectTM Advanced Glycation End Product (AGE) Competitive ELISA Kit (Cell Biolabs Inc.; cat # STA-817) as per manufacturer’s instructions.

Techniques: Isolation, Competitive ELISA, Dot Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Glycation damage to organelles and their DNA increases during maize seedling development

doi: 10.1038/s41598-022-06454-7

Figure Lengend Snippet: DJ-1 protein in maize mitochondria and plastids. The amount of DJ-1 protein from isolated mitochondria and plastids was measured using anti-DJ-1 antibodies with competitive ELISA ( A – D ) and slot blot ( E – H ) assays. For ELISA, DJ-1 protein in mitochondria was measured as ng/mL. Representative images of slot blot assays are shown ( I – L ) and the relative DJ-1 protein was determined, where the image intensity was set to 1 for leaf 1 (L1) in ( E , G ) and for light-grown leaves in ( F , H ). Uppermost panels of I-L show DJ-1 proteins, middle panels show the total protein (TP) levels in samples, and lowermost panels show mitochondrial (COX2)- and plastid (RbcL)-specific proteins (COX2 in panels I and J; RbcL in panels K and L). Cross-reactivity of maize organellar DJ-1 with anti-DJ-1 antibodies (human Park7) was confirmed and is shown in Supplementary Information, Fig. S1.

Article Snippet: AGE in plastid and mitochondrial proteins was determined using the OxiSelectTM Advanced Glycation End Product (AGE) Competitive ELISA Kit (Cell Biolabs Inc.; cat # STA-817) as per manufacturer’s instructions.

Techniques: Isolation, Competitive ELISA, Dot Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Glycation damage to organelles and their DNA increases during maize seedling development

doi: 10.1038/s41598-022-06454-7

Figure Lengend Snippet: AGE in DNA isolated from mitochondria and plastids. For competitive ELISA assays ( A – D ), AGE levels were determined as µg/mL. The relative levels of AGE were also determined by slot blot assays ( E – H ), and representative images are shown ( I – L ) where the image intensity was set to 1 for Stalk lower in ( E , G ) and for dark-grown leaves in ( F , H ). Equal amounts of mtDNA extracted from the different tissues were used in these assays. Lower panels of ( I – L ) show the equal amount of orgDNA as determined by slot blot using mitochondrial- and plastid-specific probes (cox2 and rbcL, respectively).

Article Snippet: AGE in plastid and mitochondrial proteins was determined using the OxiSelectTM Advanced Glycation End Product (AGE) Competitive ELISA Kit (Cell Biolabs Inc.; cat # STA-817) as per manufacturer’s instructions.

Techniques: Isolation, Competitive ELISA, Dot Blot

Journal: Biomedicines

Article Title: Normalizing HIF-1α Signaling Improves Cellular Glucose Metabolism and Blocks the Pathological Pathways of Hyperglycemic Damage

doi: 10.3390/biomedicines9091139

Figure Lengend Snippet: The glycolytic side-product MGO activates nuclear translocation of HIF-1α by inducing post-translational glycation and inhibition of PHD2 activity. IP of MGO-protein adducts (MGO adds) ( A ) or PHD2 ( B ) on HUVEC treated (MGO) or not (Ctr) with 200 µM MGO for 6 h, using a specific antibody for MGO modified proteins ( A ) or a specific anti-PHD2 antibody ( B ), respectively. Mouse IgG were used as control. Total cell lysates (Input) and immunoprecipitates were immunoblotted for PHD2 and MGO protein adducts ( A , B ). In the presence of oxygen, HIF-1α is rapidly hydroxylated by PHD2 to generate (Pro-OH) HIF-1α, which is degraded through the ubiquitin-proteasome pathway; proteasome inhibition with MG132 led to Pro-OH HIF-1α increase, and adding MGO prevented (Pro-OH) HIF-1α increase and led to HIF-1α nuclear translocation ( C ). Western blot analysis for (Pro-OH) HIF-1α in total extracts ( D ), and non-hydroxylated HIF-1α in nuclear extracts ( E ) from HUVEC, treated or untreated (UCtr) with the proteasome inhibitor MG132 (10µM) for 6 h, with or without MGO, in the presence or absence of Car. Bars represent mean ± SEM. Post hoc multiple comparison: *** p < 0.001 or * p < 0.05 vs. Ctr; ††† p < 0.001 or †† p < 0.01 vs. MGO.

Article Snippet: Intracellular formation of AGEs was measured by the OxiSelectTM Advanced Glycation End-Product Competitive ELISA Kit (Cell Biolabs, San Diego, CA, USA).

Techniques: Translocation Assay, Inhibition, Activity Assay, Modification, Control, Ubiquitin Proteomics, Western Blot, Comparison