Fresh 5 mM solutions of AzPt, 2-ADAPPt, and cisplatin were prepared in milli-Q water. 1 mM bovine serum albumin was incubated with 1, 2, or 5 mM AzPt, 2-ADAPPt, or cisplatin for 18 h at room temperature in the presence of 10 mM Na2PO4 (pH 7.4) and 1 mM Mg(NO3)2. Excess Pt(II) was removed by Sephadex G-25 medium (GE Life Sciences, 17003301) in laboratory prepared spin columns (Thermo Fisher, 89868).
Spin column
Manufactured by Thermo Fisher Scientific
Sourced in United States
Spin columns are a type of laboratory equipment used for the separation and purification of various biomolecules, such as DNA, RNA, and proteins. They function by providing a convenient and efficient way to isolate and concentrate specific target molecules from complex mixtures through a centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 affinity gel, by Merck Group (3 mentions)
Glycerol, by Thermo Fisher Scientific (2 mentions)
Np 40, by Thermo Fisher Scientific (2 mentions)
Agilent bioanalyzer, by Agilent Technologies (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Solid wildfire system, by Thermo Fisher Scientific (2 mentions)
Sephadex g 25 medium, by GE Healthcare (1 mentions)
Amicon ultra 4 filter, by Merck Group (1 mentions)
Elution buffer, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
B per bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Doxorubicin hydrochloride, by Merck Group (1 mentions)
Tau5 antibody, by Fortrea (1 mentions)
Captureselect iga affinity matrix, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti tcf3, by Abcam (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ziptip, by Merck Group (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
29 protocols using spin column
1
Platinum-Protein Binding Kinetics
2
IgA Purification from Milk and Plasma
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IgA was purified from IgG-depleted breast milk and plasma using peptide M resin (Invivogen) as previously described (76 (link)). Briefly, 200 μl of peptide M gel slurry was added to spin columns (Thermo Scientific). Following two washes with 500 μl of phosphate-buffered saline (PBS), IgG-depleted milk and plasma were added to the columns and incubated for 45 min at room temperature. Columns were spun and washed, and flowthrough was collected. Bound IgA was eluted with elution buffer (Thermo Scientific) and then neutralized with 120 μl of 1 M Tris-HCl, pH 9.0 (Thermo Scientific). Columns were washed and incubated overnight with PBS at 4°C, and eluted samples were run through the columns a second time. Eluted IgA fractions from both processes were combined, buffer exchanged, and then concentrated with Amicon Ultra-4 filters (Millipore). IgA concentration was determined via spectrophotometry at A280 using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). To confirm IgG depletion, samples were tested by human IgG enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Immunology Consultants, Inc.). IgA preparations with IgG concentrations below 0.07 mg/ml were considered successfully depleted. The cutoff of 0.07 mg/ml was selected as the corresponding concentration of 3× the blank optical density (OD) of the total IgG ELISA.
3
Recombinant Human Hsp90 Protein Purification
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Human
Hsp90α CTD (563–732 amino acids) recombinant protein
purification was performed as previously described.34 (link) Human Hsp90α NTD (amino acids 9–236; Addgene
22481) protein was expressed in E. coli BL21-DE3
cells.86 (link) BL21-DE3 expression strains were
grown overnight and used to inoculate LB medium at 37 °C supplemented
with 100 μg/mL ampicillin to an OD600 = 0.5–0.8,
followed by overnight induction of protein expression with 0.5 mM
isopropyl β-d -1-thiogalactopyranoside (IPTG) at 25
°C. After induction, cells were harvested by centrifugation at
5000g and lysed using B-PER bacterial protein extraction
reagent (ThermoFisher Scientific, Wesel, Germany). GST-tagged Hsp90
CTD and NTD proteins were affinity purified using spin columns (ThermoFisher
Scientific) and eluted using glutathione elution buffer. Protein aliquots
were made and supplemented with 5% glycerol and stored at −80
°C.
Hsp90α CTD (563–732 amino acids) recombinant protein
purification was performed as previously described.34 (link) Human Hsp90α NTD (amino acids 9–236; Addgene
22481) protein was expressed in E. coli BL21-DE3
cells.86 (link) BL21-DE3 expression strains were
grown overnight and used to inoculate LB medium at 37 °C supplemented
with 100 μg/mL ampicillin to an OD600 = 0.5–0.8,
followed by overnight induction of protein expression with 0.5 mM
isopropyl β-
°C. After induction, cells were harvested by centrifugation at
5000g and lysed using B-PER bacterial protein extraction
reagent (ThermoFisher Scientific, Wesel, Germany). GST-tagged Hsp90
CTD and NTD proteins were affinity purified using spin columns (ThermoFisher
Scientific) and eluted using glutathione elution buffer. Protein aliquots
were made and supplemented with 5% glycerol and stored at −80
°C.
4
Isolation and Characterization of U937 CDNs
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U937 cells were a kind gift from Associate Professor Gigi Chiu, National University of Singapore. Spin columns were purchased from ThermoScientific and were supplied with 10-μm filters attached while hydrophilic 8- and 5-μm membrane filters were purchased from Merck Millipore and used as supplied. ThermoScientific microcentrifuge was used in the cell disruption process for the production of CDNs from U937 monocytes. Doxorubicin hydrochloride was purchased from Sigma-Aldrich. HeLa and HEK293 cell lines were purchased from American Type Tissue Culture (ATTC, Manassas, VA, USA).
This research was reviewed and approved by the National University of Singapore, Institutional Review Board (NUS–IRB) for human biomedical research.
This research was reviewed and approved by the National University of Singapore, Institutional Review Board (NUS–IRB) for human biomedical research.
5
Tcf3 Interactome Identification
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Jurkat cells were harvested and lysed with IP lysis buffer containing 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40 and 5% glycerol (Thermo Fisher). Cell lysates were then incubated with anti-Tcf3 antibody-conjugated resin in spin columns (Thermo Fisher) overnight at 4 °C with rotation. The resin was then washed with IP lysis buffer two times, and proteins were eluted with elution buffer. The eluted protein samples were then separated by SDS‒PAGE, and excised gel segments were subjected to mass spectrometry analysis to identify the Tcf3 interactome.
6
Characterization of αAPF Antibody for Tau Immunoprecipitation
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The characterization and specificity of the αAPF conformation-specific antibody was published previously
[31 (link)]. For immunoprecipitation experiments, tosyl-activated magnetic Dynabeads (Dynal Biotech, Lafayette Hill, PA) were coated with 11.25 μg αAPF antibody (2.6 mg/mL) and diluted in 0.1 M borate (pH 9.5) overnight at 37°C. Beads were washed (0.2 M Tris, 0.1% bovine serum albumin [BSA], pH 8.5) and then incubated with either DLB or control patient brain homogenate with rotation at RT for 1 h. Beads were then washed three times with PBS and eluted using 0.1 M glycine (pH 2.8). The pH of each eluted fraction was adjusted using to pH 8.0 with 1 M Tris. Finally, some fractions were applied to a nitrocellulose membrane for dot blotting with the Tau-5 antibody (Covance, Princeton, NJ). Other fractions were again co-immunoprecipitated using spin columns (Cat# 26149, Thermo Scientific, Waltham, MA) coated with Tau-5 antibody to ensure the presence of tau APFs for subsequent AFM imaging.
[31 (link)]. For immunoprecipitation experiments, tosyl-activated magnetic Dynabeads (Dynal Biotech, Lafayette Hill, PA) were coated with 11.25 μg αAPF antibody (2.6 mg/mL) and diluted in 0.1 M borate (pH 9.5) overnight at 37°C. Beads were washed (0.2 M Tris, 0.1% bovine serum albumin [BSA], pH 8.5) and then incubated with either DLB or control patient brain homogenate with rotation at RT for 1 h. Beads were then washed three times with PBS and eluted using 0.1 M glycine (pH 2.8). The pH of each eluted fraction was adjusted using to pH 8.0 with 1 M Tris. Finally, some fractions were applied to a nitrocellulose membrane for dot blotting with the Tau-5 antibody (Covance, Princeton, NJ). Other fractions were again co-immunoprecipitated using spin columns (Cat# 26149, Thermo Scientific, Waltham, MA) coated with Tau-5 antibody to ensure the presence of tau APFs for subsequent AFM imaging.
7
CD2v Protein Purification and Quantification
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For CD2v protein purification, 293T cells were transfected with pCD2v-HA or pEmpty-HA control vector for 30 h. Cell lysates were harvested using the mammalian protein extraction reagent (MPER) (Thermo Scientific, Waltham, MA, USA) and incubated with anti-HA resin overnight using spin columns (Thermo Scientific, Waltham, MA, USA). Purified CD2v or purified control were eluted using HA peptides (1 mg/mL) (Thermo Scientific, Waltham, MA, USA) in Tris-buffer saline (TBS; Corning, NY, USA).
Quantification of purified CD2v was performed based on comparative densitometric analysis. Purified CD2v and purified control samples and bovine serum albumin (BSA) samples of known concentrations were run by SDS-PAGE, the gel was stained with Coomassie and the bands imaged with FluorChem R (Protein Simple, San Jose, CA, USA). Densitometric analysis was performed using ImageJ software and a standard curve was drawn. The concentration of purified CD2v obtained was approximately 3.5 ng/μL and 100 μL of CD2v preparation was used in individual experiments.
Quantification of purified CD2v was performed based on comparative densitometric analysis. Purified CD2v and purified control samples and bovine serum albumin (BSA) samples of known concentrations were run by SDS-PAGE, the gel was stained with Coomassie and the bands imaged with FluorChem R (Protein Simple, San Jose, CA, USA). Densitometric analysis was performed using ImageJ software and a standard curve was drawn. The concentration of purified CD2v obtained was approximately 3.5 ng/μL and 100 μL of CD2v preparation was used in individual experiments.
8
IgA Purification from Nasal Lavage Fluid
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IgA were purified from NLF samples using CaptureSelect™ IgA Affinity Matrix (Thermo, #194288005). Briefly, 90 μL of the matrix slurry diluted in PBS was loaded on the spin columns (Thermo, #69702) and centrifuged. 100 μL NLF was diluted 1:3 in PBS, loaded on a column and mixed on a horizontal shaker. Following 1 h incubation at room temperature, the columns were centrifuged and eluted liquid was collected and kept as unpurified fraction (here referred to as IgG Fraction). IgA-purified fraction was eluted with 400 μL of 0.1 M glycine (pH 3).
9
Co-Immunoprecipitation of GFP-Id2 Complexes
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GFP-Id2WT and GFP-Id2ΔHLH Jurkat cells were generated by lentiviral infection. HEK-293 cells were transfected with the appropriate plasmids. For Co-IP, a Pierce coimmunoprecipitation kit was used. Briefly, cells were harvested and lysed with IP lysis buffer containing 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40 and 5% glycerol (Thermo Fisher). Cell lysates were then incubated with the indicated antibody (anti-GFP, Santa Cruz; anti-Flag, CST Signaling; anti-Tcf3, Abcam)-conjugated resin in spin columns (Thermo Fisher) overnight at 4 °C with rotation. The resin was then washed with IP lysis buffer two times, and proteins were eluted with elution buffer. The eluted samples were then used for immunoblotting with appropriate antibodies. The immunoblot images were obtained in a Bio-Rad ChemiDoc Imaging System.
10
Plasma Proteome and Inflammatory Profiling
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Untargeted proteomics and targeted inflammatory profiling were performed on plasma collected at admission of 87 case-control pairs. Using mass spectrometry, proteins were identified and quantified as previously described (45 (link)). Briefly, 10 μl of plasma was depleted of the 12 most abundant proteins using spin columns (Thermo Fisher Scientific, Rockford, USA) following the manufacturer’s instructions. Labeled peptide pool samples were generated using the Tandem Mass Tag 10-plex kit (Thermo Fisher Scientific, Illinois, USA) and desalted using ZipTips (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Chromatographic separation of peptides was carried out on the Dionex Ultimate 3000 nanoflow liquid chromatography system on a 75 μm by 50 cm C18 reverse-phase analytical column and measured using a Q Exactive Orbitrap mass spectrometer coupled to the chromatography system via a nanoelectrospray ion source (Thermo Fisher Scientific, Illinois, USA).
Concentrations of 29 inflammatory mediators were quantified using a human cytokine magnetic bead assay (EMD Millipore, Burlington, Massachusetts, USA) on the Magpix with Xponent software (version 4.2; Luminex Corp) and acquired median fluorescent intensity data analyzed using the Milliplex analyst software (version 3.5.5.0 standard). Table S5 lists all cytokines assessed.
Concentrations of 29 inflammatory mediators were quantified using a human cytokine magnetic bead assay (EMD Millipore, Burlington, Massachusetts, USA) on the Magpix with Xponent software (version 4.2; Luminex Corp) and acquired median fluorescent intensity data analyzed using the Milliplex analyst software (version 3.5.5.0 standard). Table S5 lists all cytokines assessed.
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