Methods and analyses of NC protein purification and preparation have been previously described for WT and mutant NC HIV-1 (Guo et al., 2000 (link); Wu et al., 2014a (link), 1996 (link)), and WT RSV, MuLV, HTLV-1 (Stewart-Maynard et al., 2008 (link)), SIV (Post et al., 2016 (link)), FIV (Wu et al., 2014b (link)), and EIAV (Stewart-Maynard et al., 2008 (link)). NC proteins lyophilized from acetronitrile, water and trifluoroacetic acid, and containing 1 equivalent of Zn2+ per finger were dissolved in commercial D-PBS (Wisent), aliquoted and stored at −80°C until further use. Recombinant HIV-1 Gag protein purchased from Abcam (ab109969) was diluted in D-PBS (Wisent), as recommended by the manufacturer. Purified Gag was produced using Escherichia coli expression systems and purified using nickel-affinity columns, which maintain purified protein Zn2+ content and secondary structures permitting functional and structural studies of Zn2+ binding proteins (Colombo et al., 2013 ; Zhang et al., 2017 (link)).
D pbs
Manufactured by Wisent
Sourced in Canada
D-PBS is a commonly used buffer solution that maintains the physiological pH and osmotic pressure of biological samples. It is a sterile, isotonic phosphate-buffered saline solution.
Automatically generated - may contain errors
Lab products found in correlation
Ab109969, by Wisent (2 mentions)
Rneasy total rna kit, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Anti mouse, by Merck Group (1 mentions)
Halt proteinase phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
Ecl prime detection, by Cytiva (1 mentions)
Human elisa kit, by BioLegend (1 mentions)
Genechip human gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Human hnp 1 3 enzyme linked immunosorbent assay elisa reagents, by Hycult Biotech (1 mentions)
Affymetrix gene chip human gene 1.0 st, by Thermo Fisher Scientific (1 mentions)
Human gene 1.0 st, by Thermo Fisher Scientific (1 mentions)
Elisa, by Hycult Biotech (1 mentions)
Genechip, by Thermo Fisher Scientific (1 mentions)
Cell scraper, by Sarstedt (1 mentions)
Zinc sulphate, by Merck Group (1 mentions)
Goat antimouse igg fc specific alkaline phosphatase, by Merck Group (1 mentions)
Xfe24 microplate, by Agilent Technologies (1 mentions)
Seahorse xfe24 analyzer, by Agilent Technologies (1 mentions)
Seahorse xf base medium, by Agilent Technologies (1 mentions)
11 protocols using d pbs
1
Purification and Preparation of Retroviral NC Proteins
2
Purification and Preparation of Retroviral NC Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Methods and analyses of NC protein purification and preparation have been previously described for WT and mutant NC HIV-1 (Guo et al., 2000 (link); Wu et al., 2014a (link), 1996 (link)), and WT RSV, MuLV, HTLV-1 (Stewart-Maynard et al., 2008 (link)), SIV (Post et al., 2016 (link)), FIV (Wu et al., 2014b (link)), and EIAV (Stewart-Maynard et al., 2008 (link)). NC proteins lyophilized from acetronitrile, water and trifluoroacetic acid, and containing 1 equivalent of Zn2+ per finger were dissolved in commercial D-PBS (Wisent), aliquoted and stored at −80°C until further use. Recombinant HIV-1 Gag protein purchased from Abcam (ab109969) was diluted in D-PBS (Wisent), as recommended by the manufacturer. Purified Gag was produced using Escherichia coli expression systems and purified using nickel-affinity columns, which maintain purified protein Zn2+ content and secondary structures permitting functional and structural studies of Zn2+ binding proteins (Colombo et al., 2013 ; Zhang et al., 2017 (link)).
3
Evaluating Autophagy Markers in EBV-B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EBV-B cells were treated for 3 hours with PapMV nanoparticles or rapamycin, with or without pretreatment with 5 mM 3-MA. Cells were collected and washed Dulbecco’s phosphate-buffered saline (D-PBS, Wisent Bioproducts). Proteins were extracted in the presence of HALT proteinase/phosphatase inhibitors (ThermoFisher) from the above-mentioned pelleted cells and quantified by Bio-Rad protein assay. Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Membranes were incubated with rabbit an anti-human LC3B antibody (Ab; 1:3,000) (Novus Biologicals), a rabbit anti-ATG5-specific Ab (Cell Signaling) or a mouse anti-human β-actin-specific Ab (1:10,000) (Millipore). Membranes were revealed with horseradish peroxidase (HRP)-linked anti-rabbit (1:5,000) or anti-mouse (1:40,000 for β-actin) Abs (Chemicon) followed by enhanced chemiluminescence (ECL) prime detection (Amersham) on film. Densitometry was performed with ImageJ software and is reported as the ratio between densities of LC3-II over LC3-I bands or the ratio of ATG5 over β-actin bands.
4
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
D-PBS (Wisent Inc., Quebec, Canada), TRIzol® Reagent (Invitrogen™, Carlsbad, CA, USA), IsoPrep (Robbins Scientific Corporation, Sunnyvale, CA, USA), and the RNeasy total RNA kit (Qiagen, Hilden, Germany) for RNA preparation were employed in this research. Real-time quantitative- (qRT-) PCR utilized primers that were designed according to GenBank sequences and based on previous studies [25 (link), 26 (link)]. The primers were synthesized by Pacific Science Co., Ltd., Bangkok, Thailand. The human ELISA kit was purchased from BioLegend (San Diego, CA, USA). The other reagents used are described below.
5
Comprehensive Molecular Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study we used D-PBS (Wisent Inc., Quebec, Canada), TRIzol® Reagent (Invitrogen™, Carlsbad, CA, USA), IsoPrep (Robbins Scientific Corporation, Sunnyvale, CA, USA), RNeasy total RNA kit (Qiagen, Hilden, Germany), and the Affymetrix GeneChip® Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA, USA). For qRT-PCR, we designed primers based on previous studies and Genbank, and had primers synthesized by Pacific Science Co., Ltd. (Bangkok, Thailand). Human HNP 1–3 enzyme-linked immunosorbent assay (ELISA) reagents were purchased from Hycult® Biotech (Uden, The Netherlands). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA).
6
Plasma Proteomics and Transcriptomics in Hyperlipidemia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heparinized blood samples (5 ml) were collected once from healthy controls and from all patients before hyperlipidaemia treatment or coronary bypass grafting. Plasma (2 ml) was immediately collected by centrifugation of whole blood. Plasma aliquots were prepared for lipid measurement, and kept at −70 °C for the detection of plasma proteins encoded by the selected genes.
Packed blood cells were resuspended in D-PBS (Wisent Inc., Quebec, Canada) and used to isolate mononuclear cells. Approximately 2 × 106 PBMCs in TRIzol (Invitrogen) were kept at −70 °C for gene expression profiling by DNA microarray analysis using Affymetrix GeneChip® Human Gene 1.0 ST (Affymetrix).
Packed blood cells were resuspended in D-PBS (Wisent Inc., Quebec, Canada) and used to isolate mononuclear cells. Approximately 2 × 106 PBMCs in TRIzol (Invitrogen) were kept at −70 °C for gene expression profiling by DNA microarray analysis using Affymetrix GeneChip® Human Gene 1.0 ST (Affymetrix).
7
Heparinized Blood Profiling for Hyperlipidemia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heparinized blood samples (5 mL) were collected once from healthy donors and from all patients before hyperlipidemia treatment or coronary bypass grafting. Plasma (2 mL) was immediately collected by centrifugation of whole blood, and an aliquot for lipid measurement was kept at −70 °C for detection of HNP 1–3 by ELISA (Hycult Biotech). Packed blood cells were resuspended in D-PBS (Wisent Inc.) and used to isolate mononuclear cells. Approximately 2 million peripheral blood mononuclear cells (PBMCs) in TRIzol (Invitrogen™) were kept at −70 °C for gene expression profiling by DNA microarray analysis using Affymetrix GeneChip® Human Gene 1.0 ST. α-defensin expression was validated by qRT-PCR.
8
Lipid and Protein Analysis of CFBE41o- Cell Line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For lipid analysis, cell lines were seeded in 100 mm plates with 1,000,000 cells in 12 ml of media and grown overnight to 80% confluence and treated the next day. They were treated with fresh drugs every 24 h, for a total time of 72 h with 1.25 μM LAU-7b (Laurent Pharmaceuticals) and 12.5 μM zinc-sulphate (Sigma-Aldrich), as well as Triple therapy composed of 3 μM Elexacaftor (VX-445), 3 μM Tezacaftor (VX-661) and 10 nM Ivacaftor (VX-770). After completion of treatment, the cell monolayer was washed twice with warmed D-PBS (Wisent Bioproducts), gently scraped with a cell scraper (Sarstedt), and pipetted into a 1.5 ml screw cap tube filled with 1 ml of 1 mM butylated hydroxyanisole (BHA) in a chloroform/methanol solution (2:1 vol/vol) for mass spectrometry lipid analysis. For protein analysis, CFBE41o-(F508del) cell line was seeded in 60 mm plates with 500,000 cells in 6 ml of media and grown overnight to 80% confluence and treated the next day. The cells were also treated for 72 h, with the same drug concentrations as indicated above for the various treatment combinations.
9
Isolation and Purification of Glomeruli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glomeruli were isolated as described previously.39 (link)
Kidneys were extracted and longitudinally cut to separate medulla from cortex. The medulla was discarded and cortex samples were processed individually (such that one mouse represents one biological replicate). Cortices were minced using a #10 scalpel blade, incubated in 5 mL Dulbecco’s phosphate-buffered saline (D-PBS) solution (Wisent, St-Bruno, QC) containing 1 μg/mL collagenase for 30 minutes at 37°C with shaking at 30 rpm and then passed through a 100 μm sterile cell strainer with 40 mL cold D-PBS to enrich glomeruli. The flow-through was pelleted at 1000 rpm for 1 minute followed by removal of the supernatant. To lyse red blood cells, 5 to 10 mL of sterile Ack lysis buffer (0.15 M NH4Cl, 7 mM KCO3, 0.1 mM ethylenediaminetetraacetic acid [EDTA]) was added to the pellet, inverted 5 to 7 times at room temperature, and immediately spun down at 1000 rpm for 1 minute. The supernatant was removed and the glomeruli were lysed by re-suspending the pellet in 500 to 600 μL of lysis buffer, as described below.
Kidneys were extracted and longitudinally cut to separate medulla from cortex. The medulla was discarded and cortex samples were processed individually (such that one mouse represents one biological replicate). Cortices were minced using a #10 scalpel blade, incubated in 5 mL Dulbecco’s phosphate-buffered saline (D-PBS) solution (Wisent, St-Bruno, QC) containing 1 μg/mL collagenase for 30 minutes at 37°C with shaking at 30 rpm and then passed through a 100 μm sterile cell strainer with 40 mL cold D-PBS to enrich glomeruli. The flow-through was pelleted at 1000 rpm for 1 minute followed by removal of the supernatant. To lyse red blood cells, 5 to 10 mL of sterile Ack lysis buffer (0.15 M NH4Cl, 7 mM KCO3, 0.1 mM ethylenediaminetetraacetic acid [EDTA]) was added to the pellet, inverted 5 to 7 times at room temperature, and immediately spun down at 1000 rpm for 1 minute. The supernatant was removed and the glomeruli were lysed by re-suspending the pellet in 500 to 600 μL of lysis buffer, as described below.
10
Enzyme-Linked Immunospot Assay for Antigen-Specific Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect antigen‐specific responses, 96‐well ELISpot IP filter plates, 0.45 μm, clear (Millipore Canada Ltd., Etobicoke, ON, Canada) were coated with 5 μg of recombinant LG3 without the His8G‐tag or MSA diluted in Dulbecco's phosphate buffered saline (D‐PBS; Wisent Bioproducts, St‐Bruno, QC, Canada). To measure the number of total IgM and IgG‐antibody‐secreting cells, plates were coated with 1.5 μg of goat antimouse IgG (H + L) F(ab'2) (Sigma‐Aldrich, Canada Ltd., Toronto, ON, Canada). Activated cells were added for 18‐24 hours at 37°C in a 5% CO2 humidified incubator. Following washing, bound Ig were detected using goat antimouse IgG (Fc specific)‐alkaline phosphatase or goat antimouse IgM (μ specific)‐alkaline phosphatase antibodies (both from Sigma‐Aldrich). ELISpots were developed using 100 μl/well of 5‐Bromo‐4‐chloro‐3‐indolyl‐phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (MabTech, Inc, Cincinnati, OH) to visualize the spots. The reaction was stopped by tap water. The plates were read by an automated ELISpot reader and data were analyzed by ImmunoSpot Analyzer 5.1 software (Cellular Technology Limited, Cleveland, OH).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!