We used the “M” strain (ATCC BAA-535) of Mycobacterium marinum. The bacteria were grown at 30°C with slow agitation in Middlebrook 7H9 broth medium (Becton Dickinson), enriched with 0.5% albumin (SIGMA), 0.5% glycerol (SIGMA), 0.4% glucose (Biopack), and 0.25% Tween 80 (SIGMA) and was used at the mid‐ to late‐log phase (OD = 1.0). The bacteria were kindly provided to us by Professor Maria I. Colombo (IHEM-CONICET, Mendoza, Argentina) (9 (link)). Cultured bacteria were washed twice with phosphate‐buffered saline (PBS) before use. For disrupting bacterial clumps, suspensions were passed 5–10 times through a 200 μL-micropipette tip and centrifuged at 1500 rpm for 1 min. To verify the bacterial dose, M. marinum suspension was diluted in serial dilutions and plated on 7H10 agar (Becton Dickinson) enriched with 0.5% albumin (Sigma), 0.5% glycerol (Sigma) and 0.4% glucose (Biopack) for counting bacterial colonies.
Albumin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Belgium, Sao Tome and Principe, Japan, India
Albumin is a laboratory equipment product used in various clinical and research applications. It is a purified protein derived from human or bovine sources. Albumin serves as a versatile reagent and is commonly used for various purposes, including protein assays, cell culture media, and buffer formulations.
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Lab products found in correlation
Carbonic anhydrase, by Merck Group (14 mentions)
Cytochrome c, by Merck Group (11 mentions)
Alcohol dehydrogenase, by Merck Group (11 mentions)
β amylase, by Merck Group (11 mentions)
Glucose, by Merck Group (10 mentions)
Potassium chloride (kcl), by Merck Group (10 mentions)
Sodium chloride (nacl), by Merck Group (9 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Hepes, by Merck Group (7 mentions)
Ascorbic acid, by Merck Group (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dextrose, by Merck Group (6 mentions)
Oleic acid, by Merck Group (6 mentions)
Hemoglobin, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Hydrochloric acid (hcl), by Merck Group (6 mentions)
Cysteine, by Merck Group (5 mentions)
Thyroglobulin, by Merck Group (5 mentions)
Myoglobin, by Merck Group (5 mentions)
Urea, by Merck Group (5 mentions)
218 protocols using albumin
1
Mycobacterium marinum Culture and Quantification
2
Silver Nanoparticles in Albumin-PBS
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Total of 4 mg silver nanoparticles of 20 nm nominal diameter (PlasmaChem, Berlin, Germany) were suspended in 1600 mm3 of MilliQ water and sonicated (4.2 kJ/cm3) with an ultrasonic homogenizer. Then, albumin (Sigma, Darmstadt, Germany) and concentrated PBS (Sigma, Darmstadt, Germany) were added to reach 2 mg/cm3 concentration of nanoparticles in PBS containing 1% albumin.
3
Anti-glycation Effects of Mineral Extracts
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Example 1
Three mineral extract compositions were evaluated to test the anti-glycation effect.
Each reaction mixture contained 10 mg/ml albumin (Sigma) in PBS with 500 mM glucose (Sigma G8270) in PBS. The negative control was 10 mg/ml albumin without glucose. A negative control was used for each experimental point. The positive control was 10 mg/ml albumin with 500 mM glucose with 1 mM aminoguanidine hydrochloride (Sigma 396494).
Sample Preparation
Each mineral extract composition was diluted in type I sterile water to 10 mg/ml, sterilized by 0.22 micron filtration and incubated at dilutions 100 ug/ml, 10 ug/ml, 1 ug/ml and 0.1 ug/ml with the reaction mixture with or without glucose for days at 37° C. in the presence of sodium azide.
Protein glycation was detected by the increase of non-tryptophan fluorescence (excitation at 360 nm, emission at 460 nm) using microplate fluorometer Cytofluor 2350 (Millipore), as described (Argirova and Argirov, 2003). The glycation value for each experimental point was obtained by subtracting the background reading (samples without glucose).
Results and Discussion
As seen in
4
Fluorescent Mycobacterial Strains for Research
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WT M. marinum M strains (ATCC #BAA-535) expressing tdTomato, tdKatushka, or EBFP2 under the msp12 promoter (Takaki et al., 2013 (link)) were grown under hygromycin B (Formedium) selection in 7H9 Middlebrook medium (Difco) supplemented with oleic acid, albumin, dextrose, and Tween-80 (Sigma) (Takaki et al., 2013 (link)).
M. tuberculosis ΔleuD ΔpanCD double auxotroph expressing mCherry or GFP under control of the msp12 promoter were grown under hygromycin B (Formedium) and kanamycin selection in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose, catalase, Tween-80 (Sigma), 0.05 mg/ml L-leucine and 0.024 mg/ml calcium pantothenate (Sigma) (Sampson et al., 2011 (link)).
M. tuberculosis ΔleuD ΔpanCD double auxotroph expressing mCherry or GFP under control of the msp12 promoter were grown under hygromycin B (Formedium) and kanamycin selection in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose, catalase, Tween-80 (Sigma), 0.05 mg/ml L-leucine and 0.024 mg/ml calcium pantothenate (Sigma) (Sampson et al., 2011 (link)).
5
Radiolabeling of α-Synuclein and Amyloid Beta Peptides
6
Kinase activity assay of Stk and Fem proteins
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One µg recombinant proteins (FemX, FemA or FemB) were incubated with 1 µg Stk kinase domain (StkKD) in kinase buffer (50 mM Tris-HCl pH 7.5, 3 mM MgCl2, 3 mM MnCl2) with or without 20 mM ATP for 1 h at 37 °C. For the dephosphorlyation reaction, 1 µg Stp was added to the reaction mixture. Each reaction was stopped by addition of 5 x SDS sample buffer and heating for 5 min 95 °C. The phosphorylated proteins were detected by Western blotting using an anti-phosphothreonine antibody (#9381 S, Cell Signaling, Frankfurt, Germany). SDS-PAGE was performed and the proteins were blotted on a nitrocellulose membrane using a semi-dry blotting technique. The blot was blocked in 5% albumin (Sigma-Aldrich, Taufkirchen, Germany) in TBS (10 mM Tris-HCl pH 7.5, 0.9% NaCl) for 2 h and then incubated with anti-phosphothreonine antibody (1:5,000, 1% albumin/TBS-T) overnight at 4 °C. After washing the blot three times in TBS-T (TBS + 0.05% Tween 20) for 15 min, the blot was incubated with anti-rabbit-HRP antibody (#L3012, SAB, USA) (1:10,000 in TBS) for 2 h. After three additional TBS-T washing steps, the blot was incubated with ECL substrate for 2 min. Phosphorylated proteins were visualized using ImageQuant LAS 4000 imaging system (GE Healtcare, Munich, Germany).
7
Astrocyte Senescence Markers Analysis
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Astrocytes were seeded on tissue culture dishes at a density of 10,000 cells/cm2 and analyzed for senescence markers after 7 days of treatment. Cells were treated by adding either 10 nM TGFβ1 (PeproTech) or 0.2 mM albumin (Sigma–Aldrich) to fresh culture media every 48 h. For TGFβ receptor inhibition, cells were pretreated with either 1 μM SJN2511 (Tocris Biosciences) or DMSO (Sigma–Aldrich) control for 30 min prior to albumin treatment.
8
Biochemical Assays for Tryptophan, Indole, and Albumin
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Assays for Tryptophan, indole (Sigma Aldrich, St. Louis, MO) and albumin (Sigma Aldrich, St. Louis, MO) levels were performed using the commercially available kits according to manufacturer's protocol.
9
Gelatin Zymography for Proteolytic Activity
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The proteolytic activities were determined in 10% polyacrylamide gels copolymerized with 0.2% bovine gelatin (Sigma-Aldrich), holo-Lf (NutriScience Innovations, LLC, Milford, CT, USA), hemoglobin, albumin, and porcine fibrinogen (Sigma-Aldrich). The protein concentration of PpAs was adjusted to 20 µg for use in the electrophoretic run. Electrophoresis was performed at a constant voltage (100 V) for 2 h in an ice bath (4 °C). Samples were not treated with β-mercaptoethanol or boiled. After electrophoresis, the gels were washed twice with 2.5% (v/v) Triton X-100 (Sigma-Aldrich) solution for 30 min and incubated overnight at 37 °C with different buffers: 100 mM sodium acetate, 2 mM CaCl2 (pH 5.0), 100 mM Tris-2 mM CaCl2 (pH 7.0), and 100 mM Na2CO3-NaHCO3-2 mM CaCl2 (pH 9.0) (all from Sigma-Aldrich). Finally, the gels were stained with 0.5% (w/v) Coomassie brilliant blue R-250 (Bio-Rad, Feldkirchen, Germany). Protease activities were identified as clear bands on a blue background. The gels were subjected to a washing solution until the proteolytic bands were visualized. All assays were performed in three independent experiments.
10
Bisphenol and Hormone Exposure Protocol
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