AgNO3, bacterial culture materials, and catalase (CAT) and glutathione peroxidase (GPx) enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other reagents were purchased from the following companies: LDH Assay Kit (colorimetric) from Abcam (Cambridge, UK); PiBind resin from Expedeon (San Diego, USA); TRIzol reagent from Life Technologies (California, USA); Maxima SYBR Green/Fluorescein qPCR Master Mix and QuantiTects Reverse Transcription Kit from Qiagen (Germantown, USA); and TriFast from Peqlab VWR (Pennsylvania, USA).
Pibind resin
Manufactured by Abcam
Sourced in United Kingdom, United States
PiBind resin is a solid-phase affinity chromatography resin designed for the purification of proteins containing a polyhistidine tag. The resin consists of nickel-charged nitrilotriacetic acid (Ni-NTA) agarose beads that selectively bind to polyhistidine-tagged proteins, allowing for their separation and purification from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Quantitects reverse transcription kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Glutathione peroxidase, by Merck Group (2 mentions)
Maxima sybr green fluorescein qpcr master mix, by Qiagen (2 mentions)
Ldh assay kit, by Abcam (2 mentions)
Atpase assay kit, by Abcam (2 mentions)
Agno3, by Merck Group (1 mentions)
Silver nitrate agno3, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
M13mp18 ssdna, by New England Biolabs (1 mentions)
High throughput colorimetric atpase assay kit, by Abcam (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Calcineurin cellular activity assay kit, by Enzo Life Sciences (1 mentions)
Tautomycin, by Enzo Life Sciences (1 mentions)
Subcellular protein fractionation kit for tissue, by Thermo Fisher Scientific (1 mentions)
14 protocols using pibind resin
1
AgNO3 Oxidative Stress Assay
2
Fungal-Mediated Synthesis of Silver Nanoparticles
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Silver nitrate (AgNO3), fungal culture medium, and enzyme activity assay kits (colorimetric; catalase [CAT], and glutathione peroxidase [GPx] were obtained from Sigma-Aldrich (St. Louis, MO, USA), while lactate dehydrogenase [LDH]) from Abcam (Cambridge, UK). PiBind resin was purchased from Expedeon (San Diego, CA, USA); TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA); Maxima SYBR Green/Fluorescein qPCR Master Mix and the QuantiTects Reverse Transcription Kit was obtained from Qiagen (Germantown, MD, USA); and TriFast was purchased from Peqlab VWR (Radnor, PA, USA). The silver nanoparticles were previously synthesized by our team using Desertifilum sp. IPPAS B-1220 (D-SNPs) and Nostoc sp. Bahar_M (N-SNPs); their physicochemical properties were analyzed by ultraviolet–visible (UV–Vis) spectrophotometry, X-ray diffraction, Fourier-transform infrared (FTIR) spectroscopy, and scanning (SEM) and transmission electron microscopy (TEM). The D-SNP and N-SNP particle size ranged from 4.5 to 26 nm and 8.5 to 26.4 nm, respectively, with an average diameter of 14.7 ± 1.1 nm and 14.9 ± 0.5 nm, respectively [33 (link),34 (link)].
3
Enzyme Activity Evaluation Protocol
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Silver nitrate (AgNO3), bacterial culture materials, and chemicals related to evaluating enzyme activities were purchased from Sigma-Aldrich (St. Louis, MO, United States), and PiBind resin was purchased from Expedeon, San Diego, United States.
4
Comprehensive Cell Line Characterization
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The MCF-7, HCT-116, and HepG2 cells were procured from the Medical Research Institute, Alexandria, Egypt. All cell culture reagents, media, and standard chemicals were procured from Sigma–Aldrich (St. Louis, MO, USA); the LDH assay kit (Colorimetric) and all antibodies (Table 1 ) were from Abcam (Cambridge, UK); and, the PiBind resin was from Expedeon (San Diego, CA, USA).
5
Biogenic Silver Nanoparticle Synthesis
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Silver nitrate (AgNO3), bacterial culture materials, and the enzymes catalase (CAT) and glutathione peroxidase (GPx) were purchased from Sigma-Aldrich (St. Louis, MO, USA); the LDH Assay Kit (colorimetric) was purchased from Abcam (Cambridge, UK); and PiBind resin was purchased from Expedeon (San Diego, USA). TRIzol reagent was purchased from Life Technologies (California, USA), and Maximas SYBR Green/Fluorescein qPCR Master Mix and the QuantiTects Reverse Transcription Kit were purchased from Qiagen (Germantown, USA). TriFast was purchased from Peqlab VWR (Pennsylvania, USA). Spherical (8.5 to 26.44 nm; mean size 14.9 nm) biogenic SNPs were extracellularly synthesized using Nostoc Bahar M (N-SNPs), characterized and published previously (Fig. 1 ).13 (link)
6
Characterization of RecA Protein ATPase Activity
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The inteinless RadA protein and the splicing inactive FL-RadAi-AA protein precursor were Ni-NTA-purified as described above, exchanged into assay buffer (25 mM Tris pH 7.5, 10 mM MgCl2) pre-treated with PiBind resin (Innova Biosciences) to remove contaminating inorganic phosphate. ATPase activities of RadA and FL-RadAi-AA (2 μM) were analyzed using the High Throughput Colorimetric ATPase Assay kit (Innova Biosciences) following the manufacturer instructions. Enzymatic reactions were performed at different temperatures (25–85°C with 10°C increments) for 30 min. Different concentrations of the substrate, ATP, was added in the presence of 0.2 μg/ml of M13mp18 ssDNA (NEB). To eliminate the impact of temperature-dependent non-enzymatic ATP hydrolysis, protein-free controls were used for each data point. All experiments were performed in triplicate.
7
Corneal Endothelial-Descemet Membrane ATPase Assay
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Two corneal endothelial-Descemet membrane (CEDM) peelings from the same mouse were pooled and homogenized in 30 µL assay buffer, provided in the ATPase Assay kit (Abcam #ab234055), using a plastic disposable pestle. After sonication, the sample was centrifugated at 10,000× g for 10 min at 4 °C. The supernatant was recovered, and the phosphates in the sample were depleted by incubation in 40 µL of PiBind resin (Innova Biosciences #501-0015, Montluçon, France) for 15 min at room temperature in a rotary device. After centrifugation at 1000× g for two minutes, the sample was recovered, and ATPase activity was measured in 5 µL of sample in the presence or absence of 1 mM ouabain. Na+-K+ ATPase activity (n ≥ 3 for Ctrl and Tm) was obtained by subtracting the activity in presence of ouabain from the total activity. Protein was measured using the BCA method.
8
GTPase Activity Assay for P. gingivalis Ndk
9
Na+/K+ ATPase Activity Assay
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Six CEDM (6 mice) were pooled and homogenized in 130 µl of assay buffer provided by Na+/K+-ATPase activity kit (#K417-100, BioVision) using plastic disposable pestle. The sample was sonicated and then centrifuged at 10,000 g for 10 minutes. The supernatant was recovered and phosphates in the sample were depleted by incubating with 40 µl of PiBind resin (#501-0015, Innova Biosciences) for 15 minutes at room temperature in a rotary device. After centrifugation at 1,000 g for two minutes the sample was recovered, and ATPase activity was measured in 5 µl of sample in the presence or absence of 1 mM ouabain. Na+/K+-ATPase activity was obtained by subtracting the activity in presence of ouabain from the total activity. Protein was measured by BCA method.
10
ATP Hydrolysis Quantification in Astrocytes
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Whole-cell lysates were prepared from the primary cortical astrocytes as described above for the ATP quantification. Contamination with inorganic phosphate (Pi) was removed via incubation of the lysate with Pi Bind resin (Innova Biosciences) for 2 h at +4°C. ATPase activity was quantified in aliquots of 10 μg of protein using ATPase assay kit (Innova Biosciences) according to the manufacturer’s instructions. The amount of Pi released was quantified colorimetrically at 630 nm using Infinite M1000 Pro multi-mode microplate reader (Tecan). Pi standard curve was established in each experiment.
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