Purification was performed on the MBPTrap HP 5 ml column (Cytiva, United States) using ÄKTA Pure (GE Healthcare, United States). Cell paste which was obtained following centrifugation of 500 ml of culture was resuspended in 30 ml binding buffer (20 mM Tris–HCl, 200 mM NaCl, and pH 7.4) and was sonicated until the solution was clear. The supernatant was obtained by centrifugation at 13,000 rpm for 40 min and purified using the MBPTrap HP column which had been equilibrated with binding buffer (pH 7.4). PMM/PGM was eluted from the column by washing with binding buffer supplemented with 20 mM maltose. The eluted protein was further purified with a HiLoad™ 16/60 Superdex™ 200 pg. column (GE Healthcare, United States), which had been pre-equilibrated with buffer containing 20 mM Tris–HCl (pH 7.4), and 150 mM NaCl. Fractions containing PMM/PGM were identified by SDS-PAGE. The concentrations of the protein fractions were measured by UV–Vis Spectrophotometer (DeNovix, United States).
Uv vis spectrophotometer
Manufactured by DeNovix
Sourced in United States
The UV-Vis Spectrophotometer is a laboratory instrument that measures the absorption or transmission of light in the ultraviolet and visible wavelength range. It is used to quantify the concentration of a sample by analyzing its light absorption characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Dna rna shield fecal collection tube, by Zymo Research (2 mentions)
Zymobiomics dna miniprep kit, by Zymo Research (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Kta pure, by GE Healthcare (1 mentions)
Quick dna fecal soil microbe miniprep kit, by Zymo Research (1 mentions)
Trireagent, by MP Biomedicals (1 mentions)
Revertaid first strand cdna synthesis kit with oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
6 protocols using uv vis spectrophotometer
1
Purification of Phosphomannomutase/Phosphoglucomutase
2
Fecal DNA Extraction and Microbiome Analysis
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Collecting feces samples was undertaken by the standard operating procedures (SOPs) based on the International Human Microbiome Standard (IHMS) protocol (Costea et al. 2017 (link)). Within 2 weeks of fecal sample collection, participants were asked to discontinue antibiotics, prebiotics, probiotic supplements, or proton pump inhibitors (PPIs). Fecal samples were stored in DNA/RNA Shield™ Fecal Collection Tubes (Zymo Research Corp., Irvine, CA, USA) at − 80 °C until fecal DNA extraction. The ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corp., Irvine, CA, USA) was applied to extract total DNA from fecal samples as indicated in the manufacturer’s recommendation. The quality and concentration of DNA were assessed by using a DeNovix UV–Vis spectrophotometer (DeNovix, Wilmington, DE, USA), and the specimens were kept at − 20 °C for subsequent analysis.
3
Quantitative Analysis of FBXW7 and SREBP1a
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At 48 h after transfection, the cells were collected to extract total RNA using the TRIzol™ Plus RNA Purification Kit (CW0580S; CWBIO, Cambridge, MA, USA). The purity of RNA (OD260/OD280) was determined using a UV-Vis spectrophotometer (DeNovix, Wilmington, DE, USA). Complementary DNA (cDNA) was synthesized by a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Thereafter, fluorescence quantitative polymerase chain reaction (qPCR) was carried out using the following reaction system: RNase free dH2O 8.2 µL, cDNA 1 µL, upstream primer 0.4 µL, downstream primer 0.4 µL, and universal SYBR qPCR Master Mix 10 µL. The reaction steps were as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10 s; annealing at 58 ℃ for 30 s; and extension at 72 ℃ for 30 s, for a total of 40 cycles. The primer sequence was obtained from General Biosystems Co., Ltd (Anhui, China), and was as follows: FBXW7 (F: 5'-AGCTGTCCAGCCCCTTCTA-3'; R: 5'-GCACGCTTGTGATTCTCCTT-3'); SREBP1a (F: 5'-GCCTATTTGACCCACCCTAT-3'; R: 5'-TGGCACTGACTCTTCCTTGA-3'); β-actin (F: 5'-TGGCACCCAGCACAATGAA-3'; R: 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'). The relative expression levels of FBXW7 and SREBP1a were calculated by 2-∆CT method and normalized to β-actin.
4
Fecal DNA Extraction and Quantification
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All participants provided fecal samples in DNA/RNA Shield™-Fecal Collection tubes (Zymo Research Corp, Irvine, CA, USA), which were stored at −80 °C until further analysis. The fecal samples were extracted for DNA using the ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corp, Irvine, CA, USA) according to the manufacturer’s instructions. The quality of DNA and its concentration were identified using a DeNovix™ UV-Vis spectrophotometer (DeNovix Inc, Wilmington, DE, USA).
5
Fecal Microbiome DNA Isolation
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Patients were asked to stop antibiotics, prebiotics and probiotics supplement, or proton pump inhibitors (PPIs) within 2 weeks before acceptance and during the study period. Participants collected fecal samples at into tubes with DNA stabilizer (DNA/RNA Shield™ Fecal Collection Tube) and kept at − 80 °C until further microbiota analyses. The procedure was done following the standard protocol from the International Human Microbiome Standard (IHMS)8 (link). Total microbial DNA from feces were extracted using Quick-DNA™ Fecal/Soil Microbe Miniprep Kit (Zymo Research Corp.) according to the manufacturer’s protocol. DNA concentration and purity was measured by DeNovix™ UV–Vis spectrophotometer and will be stored at − 20 °C until perform the sequencing.
6
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the tissues using TRI Reagent (Sigma-Aldrich) as per the manufacturer’s instructions. Tissue sample of 30–50 mg was homogenized in 1 ml of TRI Reagent using lysing matrix D beads (1.4 mm) in FastPrep homogenization system (MP Biomedicals). RNA pellet was dissolved in diethyl pyrocarbonate (DEPC) treated nuclease-free water and stored at –80 °C until further use. RNA concentration and purity were analysed in Denovix UV-V is spectrophotometer (Denovix). To remove the residual genomic DNA, 1 µg total RNA was treated with 1U of DNase I, RNase free (Thermo Scientific) following manufacturer’s protocol. DNase-treated total RNA (1 µg) was reverse-transcribed using RevertAid First Strand cDNA Synthesis kit with oligo-dT primer (Thermo Scientific) as per the manufacturer’s instructions.
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