A membrane protein detergent screen on AmtB-GFP was performed as previously described16 (link) with minor modifications. Briefly, AmtB-GFP was extracted from purified membranes (described above) with 1-2% (w/v) of the detergent of interest in Buffer B and incubated for one to three hours at room temperature or overnight at 4 °C with gentle agitation. Insoluble material was pelleted by centrifugation at 20,000 g for 25 minutes at 4 °C. The clarified supernatant was loaded onto small Ni-NTA agarose (Qiagen) drip columns (Bio-spin Chromatography columns, Bio-Rad) and washed with several column volumes of Buffer C (200 mM sodium chloride, 20 mM imidazole, 5 mM BME, 50 mM Tris, pH 8.0 at room temperature) supplemented with two times the critical micelle concentration (CMC) of the detergent of interest. AmtB-GFP was eluted with two column volumes of Buffer D (100 mM sodium chloride, 250 mM imidazole, 5 mM BME, 2x CMC detergent of interest, and 50 mM Tris, pH 8.0 at room temperature). Eluted protein was concentrated using a 100 kDa Molecular Weight Cutoff (MWCO) concentrator and buffer exchanged into MS Buffer (2x CMC detergent of interest and 200 mM ammonium acetate, pH 8.0 with ammonium hydroxide) using a centrifugal buffer exchange device (Micro Bio-Spin 6, Bio-Rad).
Micro bio spin 6
Manufactured by Bio-Rad
Sourced in United States, Canada
The Micro Bio-Spin 6 is a disposable spin column designed for rapid purification of biomolecules such as DNA, RNA, and proteins. It utilizes a size-exclusion chromatography principle to separate target analytes from unwanted contaminants or salts. The column is pre-packed with a proprietary gel matrix and can be used for a variety of sample preparation applications.
Automatically generated - may contain errors
Lab products found in correlation
Bio spin chromatography column, by Bio-Rad (2 mentions)
Small ni nta agarose, by Qiagen (2 mentions)
Q exactive uhmr hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Bovine ubiquitin, by Merck Group (2 mentions)
Superdex 200 10 300 gl, by GE Healthcare (2 mentions)
Microrna spike in kit, by Agilent Technologies (2 mentions)
Agilent microarray scanner, by Agilent Technologies (2 mentions)
Ammonium acetate, by Merck Group (2 mentions)
Mirna complete labeling and hyb kit, by Agilent Technologies (1 mentions)
Calf intestine alkaline phosphatase, by GE Healthcare (1 mentions)
Gene expression wash buffer 1, by Agilent Technologies (1 mentions)
Agilent genespring gx, by Agilent Technologies (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Synapt g2 hdms mass spectrometer, by Waters Corporation (1 mentions)
Bovine cytochrome c, by Merck Group (1 mentions)
Ammonium acetate, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Equine myoglobin, by Merck Group (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
33 protocols using micro bio spin 6
1
Membrane Protein Detergent Screening
2
Profiling microRNA Expression Patterns
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Analysis of microRNA expression profile were performed with the same samples used for mRNA microarray, using the commercially available Agilent Unrestricted Human miRNA Microarray (V3) 8 × 15K slides Release 12.0 (Agilent Technologies, G4471A-021827) and miRNA Complete Labeling and Hyb Kit (Agilent Technologies, 5190–0456), according to the manufacturer’s protocol. Briefly, total RNA (100 ng) from each sample were dephosphorylated with Calf Intestine Alkaline Phosphatase (GE Healthcare, Amersham, UK), and linked to a Cyanine 3-labeled nucleotide (Cyanine 3-pCp) using an enzyme T4 RNA ligase (GE Healthcare, E2050Y). The labeled RNA was purified (MicroBioSpin 6, Bio-Rad, 732–6221), hybridized (overnight for 20 hours at 55 °C and 20 rpm) and washed (Agilent, 5188–5327) before scanning.
3
Native MS Analysis of Membrane Proteins
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Membrane protein samples were buffer-exchanged into aqueous ammonium acetate (200 mM, pH 7.4 adjusted with ammonium hydroxide) supplemented with 0.5% C8E4 (AA C8E4) for AmtB or 0.065% C10E5 (AA C10E5) for the other proteins using a centrifugal desalting column (Micro Bio-Spin 6, Bio-Rad). The protein–lipid mixtures were loaded into a gold-coated glass emitter (prepared in-house) and introduced into a Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo). The instrument parameters were set as follows: For AmtB, capillary voltage of 1.50 kV; capillary temperature of 200 °C; Collision-Induced Dissociation (CID) of 50 V; Collision Energy (CE) of 80 V; trapping gas pressure setting to 3.0; source DC offset of 20 V; injection flatapole DC of 10 V; inter flatapole lens of 6 V; Bent flatapole DC of 4 V; transfer multipole DC of 6 V. For TRAAK and TREK2, capillary voltage of 1.50 kV; capillary temperature set to 300 °C; CID of 50 V; CE of 50 V; trapping gas pressure set to 5.0; source DC offset of 40 V; injection flatapole DC of 8 V; inter flatapole lens of 4 V; bent flatapole DC of 3 V; transfer multipole DC of 3 V. The MS data collected from native MS were deconvoluted using UniDec and Protein Metric software.43,44 (link) Mass error was determined for the bound lipids based on the center of each peak.45 (link)
4
Mouse miRNA Expression Profiling
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Labeled miRNAs were purified on chromatography columns (Micro Biospin 6, Biorad Laboratories, Hercules, CA, USA) and then hybridized on a microarray. Each slide contains eight identical microarrays containing probes for 567 mouse and 73 mouse viral miRNAs. Hybridizations were performed at 55 °C for 20 h in a rotating oven. The hybridized microarrays were disassembled at room temperature in Agilent Gene Expression Wash Buffer 1. After the disassembly, the microarrays were washed in Gene expression Buffer 1 for five minutes at room temperature, followed by washing with Gene Expression Wash Buffer 2 for five minutes at 37 °C. The microarrays were then treated with Acetonitril for five minutes at room temperature. Post-hybridization image acquisition was performed using the Agilent scanner G2564B, which was equipped with two lasers (532 nm and 635 nm) and a 48 slides auto-sampler carousel. Data extraction from the images was accomplished by Agilent Feature Extraction 9.5 software using the standard Agilent one-color miRNA expression extraction protocol. Data analyses were performed using Agilent GeneSpring GX (version 11, Agilent Technologies, Santa Clara, CA, USA), MultiExperimentViewer (TIGR) and Microsoft Excel.
5
Non-denaturing Protein Mass Spectrometry
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ESI mass spectra were obtained with a SYNAPT G2 HDMS mass spectrometer equipped with a nanoelectrospray (nanoESI) ion source (Waters, Milford). Prior to nanoESI-MS, the protein solutions (20 mM HEPES, 100 mM NaCl) were replaced with 200 mM ammonium acetate by gel filtration with Micro Bio-Spin 6 (BioRad, Hercules) or dialysis. For obtaining the mass spectra under non-denaturing conditions, 5‒20 μM protein samples in 200 mM ammonium acetate were subjected to nanoESI-MS. For obtaining the mass spectra under denaturing conditions, the protein samples in 200 mM ammonium acetate were mixed with formic acid and methanol, resulting in 2‒3.5 μM proteins in 100 mM ammonium acetate containing 2‒3% formic acid and 30‒40% methanol. The pH of the protein solutions under the denaturing conditions was confirmed by pH test paper to be pH~1. A few microliters of the sample solution were deposited in a nanoESI emitter (HUMANIX, Japan). The parameters used for the measurement were as follows: ion source temperature 70 °C; capillary voltage 0.7‒0.85 kV; sampling cone voltage 25‒40 V. Spectra were obtained by acquiring the data for 2 min in the mass range of m/z 500‒4000 (denaturing conditions) or 1000‒4000 (non-denaturing conditions). Mass spectra were processed using MassLynx 4.2 (Waters).
6
Protein Sample Preparation and Characterization
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Methanol (> 99.9% purity) was purchased from Sigma-Aldrich (UK). Ammonium acetate was supplied by Fisher Scientific (Loughborough, UK). Ultrapure water was obtained from a Milli-Q Advantage A10 ultrapure water filtration system (Merck Millipore, Darmstadt, Germany).
Bovine ubiquitin, equine myoglobin and bovine cytochrome c were purchased from Sigma-Aldrich (UK) as lyophilized powders with purities of ≥ 98, 90 and 95% respectively. Proteins were dissolved in 200 mM Ammonium acetate. Myoglobin in Ammonium acetate was desalted twice using Micro Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Hercules, CA, US).
All samples were diluted to a final protein concentration of 10 μM.
Bovine ubiquitin, equine myoglobin and bovine cytochrome c were purchased from Sigma-Aldrich (UK) as lyophilized powders with purities of ≥ 98, 90 and 95% respectively. Proteins were dissolved in 200 mM Ammonium acetate. Myoglobin in Ammonium acetate was desalted twice using Micro Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Hercules, CA, US).
All samples were diluted to a final protein concentration of 10 μM.
7
Detergent-Exchanged Membrane Protein Preparation
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Either flash frozen samples thawed on ice or fresh samples were detergent exchanged by gel filtration chromatography. Membrane protein samples were injected onto a Superdex 200 GL 10/300 (GE Healthcare) column equilibrated in Buffer H (130 mM sodium chloride, 10% glycerol, and 50 mM Tris, pH 7.4 at room temperature) supplemented with either 0.5% of C8E4, 0.4% of NG, 0.116% OGNG, or 0.025% of DDM. Peak fractions containing the detergent-exchanged membrane protein complex were concentrated. A 50 kDa MWCO concentrator was used to concentrate samples in the detergent C8E4 and 100 kDa MWCO concentrator used on membrane protein complexes in all other detergents. Concentrated proteins were either used directly or flash-frozen in liquid nitrogen and stored at −80 °C. Notably, we found no observable difference in mass spectra quality after a single freeze-thaw of membrane proteins throughout our purification regime. Purified membrane proteins were buffer exchanged into MS Buffer (two times the CMC of detergent of interest and 200 mM ammonium acetate, pH 7.2-8.0 with ammonium hydroxide) using a centrifugal buffer exchange device (Micro Bio-Spin 6, Bio-Rad) as previously described16 (link). Membrane proteins detergent exchanged into MS Buffer supplemented with C8E4 or OGNG could be flash frozen without compromising mass spectra quality unlike MS Buffer supplemented with NG or DDM.
8
Purification and Buffer Exchange of Protein Complexes
9
Protein Sample Preparation and Purification
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Myoglobin, β-lactoglobulin, concanavalin A, carbonic anhydrase, alcohol dehydrogenase, albumin, bovine serum albumin, and transferrin were acquired from Sigma-Aldrich. Two protein components of anthrax lethal toxin, protective antigen, PA63, and the N-terminal domain of lethal factor, LFN, were graciously provided by Dr Bryan Krantz at the University of Maryland. Lyophilized proteins were reconstituted in ultrapure (18 MΩ) water. Protein samples were exchanged into either 200 mM ammonium acetate 10 mM ammonium bicarbonate pH 8 (PA63) or 200 mM ammonium acetate pH 7 (all other proteins) using Micro Bio-Spin 6 desalting columns (BioRad).
10
MicroRNA Expression Profiling in Neuroblastoma
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We performed miRNA expression profiling starting from 100 ng of total RNA. MiRNAs were labeled according to Microarray System protocol v.2.4 (Agilent Technologies). Briefly, Labeling Spike-In were added to 100 ng of total RNA and then dephosphorylated and labeled using microRNA Complete Labeling and Hyb Kit. Samples were then purified using Micro Bio-Spin 6 (Bio-Rad). After adding Hyb Spike-In, each sample was hybridized at 55°C for 20 hours on Human microRNA Microarray G4471A-029297 (Agilent Technologies), containing probes for 866 human miRNAs. After washing, slides were scanned by Agilent G2565CA scanner and images processed by Feature Extraction (FE) software using Agilent HD_miRNA protocol (Agilent Technologies). Raw and normalized data were deposited at National Center for Biotechnology Information Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/ ) and are accessible through GEO accession number GSE86889, containing GSE84841 and GSE86652 that refer to NB cell lines and NB samples, respectively.
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