AOPP-BSA was prepared as previously reported (18 (link)). Briefly, sterile BSA was incubated with HOCl at a 1:140 molar ratio at room temperature for 30 min by a light-free reaction, while the control albumin was incubated without HOCl. After the incubation period, both solutions were dialyzed overnight against sterile PBS to remove free HOCl. Contaminated endotoxin was removed by using Detoxi-Gel column (Thermo Scientific). The endotoxin in the samples was measured with the amebocyte lysate assay kit (Sigma-Aldrich) and was <0.25 EU/ml. The content of AOPP was determined by chloramine-T equivalents (μmol/L) as reported. In brief, 200 μl of the sample with 10-fold dilution was added to a 96-well plate and mixed with 20 μl of acetic acid. In standard wells, 200 μl of different concentration of chloramine-T solution (0–100 μmol/L) was mixed with 10 μl of 1.16 mol/L potassium iodide followed by the addition of 20 μl of acetic acid. The absorbance of the reaction mixture was immediately read by a microplate reader at 340 nm, showing that AOPP-BSA contained an AOPP content of 70.2 ± 1.71 nmol/mg protein, while BSA contained an AOPP content of 0.36 ± 0.02 nmol/mg protein.
Amebocyte lysate assay kit
Manufactured by Merck Group
Sourced in United States
The Amebocyte lysate assay kit is a laboratory product used to detect and quantify the presence of bacterial endotoxins. The kit contains reagents and materials required to perform the limulus amebocyte lysate (LAL) test, which is a widely recognized method for endotoxin detection.
Automatically generated - may contain errors
Lab products found in correlation
Naclo, by Guangzhou Chemical (3 mentions)
Detoxi gel column, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rat serum albumin, by Merck Group (1 mentions)
Cytosine arabinoside, by Merck Group (1 mentions)
Thiazolyl blue tetrazolium blue, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Apocynin, by Merck Group (1 mentions)
Rabbit anti β actin, by Abcam (1 mentions)
Fura 2 am, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Sb366791, by Merck Group (1 mentions)
Collagenase type ia, by Merck Group (1 mentions)
Dnase type 4, by Merck Group (1 mentions)
Rabbit anti trpv1, by Abcam (1 mentions)
Chloramine t, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
5 protocols using amebocyte lysate assay kit
1
Preparation and Characterization of AOPP-BSA
2
Oxidized Albumin Preparation and Characterization
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AOPPs were prepared as previously described (24 (link)). BSA was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and hypochlorous acid (HOCl) was purchased from Fluke Switzerland GmbH (Basserdorf, Switzerland). BSA solution (100 mg/ml) was combined with HOCl (200 mmol/l) at a molar ratio of 1:140 for 30 min at room temperature in the dark and in the absence of free amino acid/carbohydrates/lipids to exclude the formation of advance glycation end product-like structures. Prepared samples were dialyzed in 4°C PBS to remove free HOCl for 24 h. BSA was dissolved in PBS alone as control. All samples were passed through a Detoxi-Gel column (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to remove contaminating endotoxins. The endotoxin levels were measured using the Amebocyte lysate assay kit (Sigma Aldrich; Merck KGaA) and were demonstrated to be <0.025 EU/ml. The AOPP content was determined by measuring absorbance at 340 nm in an acidic condition after mixing 200 µl of prepared sample or chloramines-T with 20 µl of acetic acid in a 96-well plate. The sample was calibrated using chloramines-T in the presence of potassium iodide (14 (link)).
3
Investigating Oxidative Stress Pathways
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The following chemicals and antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA): Complete Freund's adjuvant (CFA), rat serum albumin (RSA), hypochlorous acid (HOCl), amebocyte lysate assay kit, chloramine-T, acetic acid, 4% paraformaldehyde, bovine serum albumin (BSA), trypsin, collagenase type IA, DNase type IV, cytosine arabinoside, thiazolyl blue tetrazolium blue (MTT), dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors, Fura-2/AM, apocynin, N-acetyl-l -cysteine (NAC), and SB366791. Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/L glucose), fetal bovine serum (FBS), penicillin, streptomycin and glutamine were purchased from Gibco (CA, USA). Detoxi-Gel column and BCA Protein Assay Kit were purchased from Thermo Fisher Scientific (MA, USA).
The primary antibodies used in this study were rabbit anti-NADPH oxidase 1, anti-NADPH oxidase 2, anti-NADPH oxidase 4, rabbit anti-TRPV1, rabbit anti-calcitonin gene-related peptide (CGRP) and chicken anti-beta III Tubulin antibody and rabbit anti-β-actin from Abcam (MA, USA). Secondary antibodies conjugated with FITC or Cy3 were purchased from Abcam (MA, USA).
The primary antibodies used in this study were rabbit anti-NADPH oxidase 1, anti-NADPH oxidase 2, anti-NADPH oxidase 4, rabbit anti-TRPV1, rabbit anti-calcitonin gene-related peptide (CGRP) and chicken anti-beta III Tubulin antibody and rabbit anti-β-actin from Abcam (MA, USA). Secondary antibodies conjugated with FITC or Cy3 were purchased from Abcam (MA, USA).
4
In Vitro Preparation of AOPPs
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AOPPs were prepared in vitro by incubation of RSA (Sigma-Aldrich, USA) with 40 mM NaClO (Guangzhou Chemical Reagent Factory, Guangzhou, China) in the absence of free carbohydrates/amino acid/lipids to exclude the formation of advanced glycation end product-like structures as description (Guo et al., 2008 (link)). Prepared samples were dialyzed against phosphate-buffered saline (PBS) to remove hypochlorous acid and passed through a Detoxi-Gel column (Pierce, USA) to remove contaminated endotoxin. The level of endotoxin in the samples was measured with the amebocyte lysate assay kit (Sigma-Aldrich, USA) and were found to be below 0.025 EU/ml.
AOPPs content in the preparation, which was determined by the absorbance of the mixture of samples and acetic, was 66.1±4.7 mmol/mg protein. The components of advanced glycation end products were undetected in the prepared samples.
AOPPs content in the preparation, which was determined by the absorbance of the mixture of samples and acetic, was 66.1±4.7 mmol/mg protein. The components of advanced glycation end products were undetected in the prepared samples.
5
In vitro Preparation of AOPP-HSA
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AOPP-human serum albumin was prepared in vitro by incubation of human serum albumin (HSA) (Sigma-Aldrich, USA) with 200 mM HOCl (Fluke, Switzerland) for 30 minutes in the absence of free amino acids/carbohydrates/lipids to exclude the formation of AGE-like structures as previously described.24 (link),41 (link)
The preparation was dialyzed against phosphate-buffered saline (PBS) at 4°C overnight to remove any endotoxin contamination, and endotoxin levels were determined with an amebocyte lysate assay kit (Sigma Chemical) and found to be below 0.025 EU/ml.
The preparation was dialyzed against phosphate-buffered saline (PBS) at 4°C overnight to remove any endotoxin contamination, and endotoxin levels were determined with an amebocyte lysate assay kit (Sigma Chemical) and found to be below 0.025 EU/ml.
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