Purpald

Manufactured by Merck Group

Sourced in Germany

Purpald is a laboratory reagent used for the detection and quantification of aldehydes. It is a colorimetric assay that produces a purple color when reacted with aldehydes, allowing for spectrophotometric measurement and analysis.

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Lab products found in correlation

6 protocols using purpald

In Vitro Cell Cytotoxicity Evaluation

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Dulbecco’s modified Eagle’s medium (DMEM) without phenol red, fetal bovine serum (FBS), penicillin/streptomycin, and trypsin (2.5%) were obtained from Gibco^® (Thermo Fisher Scientific, Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was procured from Bio Basic (Amherst, NY, USA). The antioxidant standards for the HPLC analysis together with other chemicals and buffers, such as DMSO, dichloro-dihydro-fluorescein diacetate (DCFH-DA), dihydroethidium (DHE), and Purpald^®, were obtained from Sigma-Aldrich (Merck, Darmstadt, Germany).
The anti-mouse IgG conjugated with HRP was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). All the antibodies for the Western blot experiments were obtained from Cell Signaling Technology^® (Danvers, MA, USA.). All the other reagents and chemicals were purchased from Merck^® (Darmstadt, Germany). The cell culture wares, including plates and flasks (polystyrene coated), were obtained from Nunc Cell Culture^® (Thermo Fisher Scientific, Waltham, MA, USA).

Kunchana K., Jarisarapurin W., Chularojmontri L, & Wattanapitayakul S.K. (2021). Potential Use of Amla (Phyllanthus emblica L.) Fruit Extract to Protect Skin Keratinocytes from Inflammation and Apoptosis after UVB Irradiation. Antioxidants, 10(5), 703.

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Antioxidant Assays for α-Cypermethrin and Curcumin

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α-Cypermethrin (CAS # 52315-07-8; purity ≥97%) was a kind gift from Nanjing Essence Fine-Chemical Co., Ltd. (Nanjing, China). Curcumin (LKT-C8069; CAS # 458-37-7; purity >90%) was from LKT Laboratories Inc. (St. Paul, MN, USA). Malondialdehyde bis (diethyl acetal) was from Merck KGaA (Darmstadt, Germany); 2,4-dinitrophenylhydrazine (DNPH), catalase (CAT; product no. C30), Red cell lysing Buffer Hybri-Max™ (product no. R7757), potassium periodate, iodonitrotetrazolium chloride, superoxide dismutase from bovine erythrocytes, xanthine, xanthine oxidase, and Purpald^® were from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). Hydrogen peroxide solution (35%) was purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).

Hongsibsong S., Stuetz W., Sus N., Prapamontol T., Grune T, & Frank J. (2014). Dietary exposure to continuous small doses of α-cypermethrin in the presence or absence of dietary curcumin does not induce oxidative stress in male Wistar rats. Toxicology Reports, 1, 1106-1114.

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Quantification of Bacterial Lipopolysaccharide

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MVs were quantified by measuring their lipopolysaccharide (LPS) content using the Purpald assay [36 (link)]. The standard test curve ranged from 0.05 to 0.4 mM of 2-keto-3-deoxyoctonate (Sigma-Aldrich). To 50 μl of each sample or standard, 50 μl of 32 mM NaIO₄ (Sigma-Aldrich) was added, and the culture was incubated for 25 min, followed by the addition of 50 μl of 136 mM of Purpald® (Sigma-Aldrich) reagent in 2 N NaOH. After a further 20 min of incubation, 50 μl of 64 mM NaIO₄ was added and the culture incubated for 20 min. Foam was eliminated from each well by the addition of 20 μl of 2-propanol. Absorbance (550 nm) was measured in a Modulus Microplate Multimode Reader (Turner Biosystems). LPS concentration of samples was normalized to 1 L of bacterial culture.

Baeza N, & Mercade E. (2020). Relationship Between Membrane Vesicles, Extracellular ATP and Biofilm Formation in Antarctic Gram-Negative Bacteria. Microbial Ecology, 81(3), 645-656.

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Catalase Activity Assay Protocol

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The assay was performed as previously described with certain modifications [21 (link)]. Briefly, the cell lysates were collected and mixed with the test buffer in a 96-well plate, and 35 mM H₂O₂ was added to the reaction mixture, which was then incubated at room temperature for 10 min on a shaker. Next, we added 3 mg/mL of Purpald^® (Sigma-Aldrich, St. Louis, MO, USA) in 0.5 M KOH to each well. After the chromogenic substance was generated, potassium periodate was added to stop the reaction. The catalase activity was measured by reading the absorbance at 540 nm using a microplate reader and calculated from formaldehyde standard curves.

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Preparation and Quantification of Salmonella Outer Membrane Vesicles

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Yo-Pro®-1 iodide was obtained from Invitrogen (Thermo Fisher Scientific Life Technologies, Darmstadt, Germany), and Purpald® (4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole and 4-Amino-5-hydrazino-1,2,4-triazole-3-thiol) was obtained from Sigma Aldrich (Merck KGaA, Darmstadt, Germany). The standard buffer in all experiments was 10 mM Tris–HCl and 4 mM MgCl₂, pH 7.6. Salmonella strains S. Typhimurium AroA::Tn10 iroBC::kan (LPS and vesicle preparation) and S. Typhimurium DB 7136 LT2 (phage propagation) were from our laboratory collections (Tindall et al., 2005 (link)). The hypervesiculating strain S. Typhimurium ATCC 14028 MvP2390 was provided by Prof. Dr. Michael Hensel (University Osnabrück). Preparation of LPS has been described previously (Andres et al., 2010 (link)). LPS was quantified in all OMV samples by the Purpald® assay, and the protein content of OMV was determined with bicinchoninic acid (BCA; Smith et al., 1985 (link); Lee and Tsai, 1999 (link)). PEG 8000 was purchased from Roth (Carl Roth GmbH, Karlsruhe, Germany). All other chemicals used were of analytic grade and double-distilled water was used throughout.

Stephan M.S., Broeker N.K., Saragliadis A., Roos N., Linke D, & Barbirz S. (2020). In vitro Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Salmonella Outer Membrane Vesicles. Frontiers in Microbiology, 11, 510638.

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Enzymatic Glycerol Oxidation Assay

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F101S PcAOX (13 μM) was reacted
with glycerol (205 mM) in 100 mM potassium phosphate pH 7.5 containing
2 mM 1,4-dithiothreitol and 1014 U catalase (Micrococcus lysodeikticus, Sigma–Aldrich), at 25 °C and 50 rpm (Innova 40 incubator
shaker; 2 mL reactions in 20 mL vials). After 68 h of incubation,
200 μL of the reactions were mixed with 200 μL of Purpald
(Sigma–Aldrich, 5 mg/mL in 0.5 N NaOH). Next, samples were
incubated at 30 °C and 700 rpm for 30 min (Eppendorf ThermoMixer
C) and subsequently mixed with 600 μL of water. Finally, 50
μL of the resulting solutions were diluted with 950 μL
of water to record their absorbance spectrum. In parallel, the same
procedure was performed for control reactions without F101S PcAOX,
except that the last dilution (1:20) was not performed before measuring.
Similarly, 200 μL of 1 mM glyceraldehyde, glycerol, and glyceric
acid were reacted with 200 μL of Purpald. Subsequently, these
standard solutions were diluted with water (final concentration =
0.2 mM, 1 mL) just prior to recording their absorbance spectrum.

Nguyen Q.T., Romero E., Dijkman W.P., de Vasconcellos S.P., Binda C., Mattevi A, & Fraaije M.W. (2018). Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol. Biochemistry, 57(43), 6209-6218.

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