The preparation of AOPPs has been described previously [5 (link), 6 (link)]. Briefly, mouse serum albumin (MSA, Sigma, St. Louis, MO, USA) solution (20 mg/ml) was incubated with 40 mM hypochlorous acid (Fluke, Buchs, Switzerland) at room temperature in phosphate-buffered saline (PBS, pH = 7.4) for 30 min. To remove the free hypochlorous acid, the prepared samples were dialyzed against PBS at 4 °C for 12 h. Finally, all samples were passed through a Detoxin-Gel™ Endotoxin Removing Gel (Pierce, Rockford, IL) to remove contaminated endotoxin. The endotoxin levels in AOPPs–MSA and unmodified MSA were measured by Limulus Amoebocyte Lysate kit (Sigma, St Louis, MO) and were found to be below 0.05 ng/mg. The concentration of prepared AOPP was detected as previously described [5 (link), 6 (link)].
Limulus amoebocyte lysate kit
Manufactured by Merck Group
Sourced in Macao
The Limulus Amoebocyte Lysate (LAL) kit is a laboratory product used to detect and quantify the presence of endotoxins, which are lipopolysaccharides derived from Gram-negative bacteria. The kit utilizes the amoebocyte (blood cell) lysate extracted from the horseshoe crab (Limulus polyphemus) to react with endotoxins, resulting in a measurable response that can be used to assess endotoxin levels in various samples.
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Lab products found in correlation
3 protocols using limulus amoebocyte lysate kit
1
Preparation of Advanced Oxidation Protein Products
2
Preparation and Characterization of Hypochlorite-Modified Albumin
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Hypochlorite modified rat serum albumin (HOCl-RSA) was prepared in vitro per our previous method20 by incubation of fatty acid-free RSA (100 g/l; Sigma-Aldrich, St. Louis, MO) with an equivalent volume of hypochlorous acid (HOCl, 200 mmol/l) or phosphate-buffered saline (PBS) for 30 min at room temperature. To prepare hypochlorite modified mouse serum albumin (HOCl-MSA), fatty acid-free MSA instead of RSA was incubated with HOCl or PBS. Prepared samples were dialysed in PBS at 4 °C overnight to remove free HOCl and then passed through a Detoxi-Gel (Thermo, Waltham, MA) to remove endotoxin contaminants.
The concentrations of endotoxin in the preparation were measured with the Limulus Amoebocyte Lysate kit (Sigma-Aldrich) and found to be <0.025 EU/ml. The content of HOCl-alb in the preparations was measured by monitoring the absorbance at 340 nm using a microplate reader under acetic acid conditions and calibrated with chloramine-T equivalents. The HOCl-alb contents in the HOCl-RSA/HOCl-MSA and unmodified RSA/MSA preparations were 6.35 ± 0.40/7.87 ± 0.39 μmol/g and 0.14 ± 0.050/0.22 ± 0.016 μmol/g protein, respectively.
The concentrations of endotoxin in the preparation were measured with the Limulus Amoebocyte Lysate kit (Sigma-Aldrich) and found to be <0.025 EU/ml. The content of HOCl-alb in the preparations was measured by monitoring the absorbance at 340 nm using a microplate reader under acetic acid conditions and calibrated with chloramine-T equivalents. The HOCl-alb contents in the HOCl-RSA/HOCl-MSA and unmodified RSA/MSA preparations were 6.35 ± 0.40/7.87 ± 0.39 μmol/g and 0.14 ± 0.050/0.22 ± 0.016 μmol/g protein, respectively.
3
Preparation of Advanced Oxidation Protein Products
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AOPPs were prepared according to the procedure described previously (Witko‐Sarsat et al., 1996 ). In brief, rat serum albumin (RSA, Sigma, St. Louis, MO, USA) solution (20 mg/ml) was incubated with 40 mm hypochlorous acid (Fluke, Buchs, Switzerland) in phosphate‐buffered saline (PBS, pH = 7.4) for 30 min at the room temperature. Prepared samples were dialyzed against PBS to remove free hypochlorous acid. To remove contaminated endotoxin, all samples were passed through a Detoxi‐Gel column (Pierce, Rockford, IL). Endotoxin levels in AOPP–RSA and unmodified RSA were then measured using a Limulus Amoebocyte Lysate kit (Sigma, St Louis, MO) and were found to be below 0.05 ng/mg protein. AOPP content in the sample was determined as described previously (Witko‐Sarsat et al., 1998 ).
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