Actin was fluorescently labeled on the surface lysine-328, using Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 647 NHS ester (Thermo Fisher Scientific), or ATTO 643 NHS ester (Atto-tec) as described in detail in (57 (link)). The Arp2/3 complex was fluorescently labeled using Alexa Fluor 488 or Alexa Fluor 568 C5-maleimide (Thermo Fisher Scientific). The protein solution was prepared for labeling by performing a buffer exchange to remove dithiothreitol (DTT) from the solution. This was accomplished by passing the protein solution through a MicroBiospin 6 column (Bio-Rad). The exchange buffer contained 20 mM Hepes at pH 7.2, 0.2 mM MgCl2, and 0.2 mM ATP. The buffer exchange was performed by centrifuging the sample at 1000g for 4 min. Next, a 10-fold excess of Alexa Fluor 488 (or Alexa Fluor 568) C5-maleimide dissolved in dimethyl sulfoxide (DMSO) was added to the Arp2/3 complex solution and incubated on ice for 1 hour. The reaction was stopped by adding 1 mM DTT to the solution. To remove any unreacted excess dye, a MicroBiospin 6 column (Bio-Rad) was used by centrifugation at 1000g for 4 min. We obtained on average 3.5 Alexa dyes per the Arp2/3 complex.
Micro bio spin 6 column
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Germany
The Micro Bio-Spin 6 columns are small, disposable purification columns designed for the rapid desalting or buffer exchange of small-volume samples. The columns contain a size-exclusion matrix that allows the separation of molecules based on their size, effectively removing unwanted salts, small molecules, or other contaminants from the sample.
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Lab products found in correlation
Masslynx software, by Waters Corporation (7 mentions)
Synapt g2 si hdms mass spectrometer, by Waters Corporation (5 mentions)
γ 32p atp, by PerkinElmer (4 mentions)
Feature extraction software, by Agilent Technologies (4 mentions)
Agilent hybridization oven, by Agilent Technologies (3 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (2 mentions)
Exactive plus emr, by Thermo Fisher Scientific (2 mentions)
Bira500, by Avidity (2 mentions)
Mirna complete labeling and hyb kit, by Agilent Technologies (2 mentions)
Unlabeled dntps, by New England Biolabs (2 mentions)
Sep pak c18 column, by Waters Corporation (2 mentions)
T4 rna ligase, by GE Healthcare (2 mentions)
Pcp cy3, by Agilent Technologies (2 mentions)
Human mirna microarray v3 kit, by Agilent Technologies (2 mentions)
Genespring gx, by Agilent Technologies (2 mentions)
Mirna labeling reagent and hybridization kit, by Agilent Technologies (2 mentions)
Agilent mirna microarray 15 k, by Agilent Technologies (2 mentions)
Amicon centrifugal filter unit, by Merck Group (2 mentions)
Dylight 488, by Thermo Fisher Scientific (2 mentions)
Lct mass spectrometer, by Waters Corporation (2 mentions)
141 protocols using micro bio spin 6 column
1
Fluorescent Labeling of Actin and Arp2/3
2
Native ESI-MS Analysis of Macromolecular Complexes
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We carried out native electrospray ionization mass spectrometry (ESI-MS) studies to decipher the macromolecular composition of TtARS1 and HvASR1, i.e. to assess whether they are monomers or dimers. As a control, we first measured ADH to confirm that the MS parameters used were suitable to maintain non-covalent complexes. Prior to analysis, all proteins were buffer-exchanged into 250 mM ammonium acetate, pH 8.0 using Micro Bio-SpinTM 6 Columns (Bio-Rad). All proteins were infused at a concentration of 15 μM. Native ESI-MS was performed on a Synapt G2-Si mass spectrometer (Waters). The capillary voltage was set to 1.8–2.1 kV, the source temperature was 30 °C, the sampling cone was set to 150 V, the source offset to 150 V, and the trap gas flow to 4 mL/min. The latter was adjusted to 5 mL/min for ADH.
3
In Vitro NME1 CoAlation Assay
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Purified recombinant His-hNME1 WT and C109A mutant were reduced with 20 mM DTT for 30 min at 25 °C. Micro Bio-SpinTM6 columns (Bio-Rad) were used to remove excess DTT. NME1 CoAlation assay was performed in nitrogen (N2)-flushed assay buffer composed of 20 mM Tris-HCl pH 8.0, and 100 mM NaCl. Reduced His-hNME1 WT or C109A (100 μM) was incubated with CoA (400 μM) in the presence and absence of CoA dimer (CoASSCoA, 400 μM) for 1.5 h at 25 °C. For oxidising conditions, recombinant proteins were incubated with CoA (700 μM) for 5 min and a further 1 h and 25 min in the presence of H2O2 (2 mM). The reactions were stopped by passing the reaction mixture through a Micro Bio-Spin™ 6 column to remove excess CoA, CoASSCoA or H2O2. NME1 CoAlation was confirmed by Western blotting with anti-CoA antibodies. Samples were used in further NDPK activity assays. For Western blotting or Coomassie-stain analysis of in vitro NME1 CoAlation, samples were incubated with 100 mM NEM for 10 min at 25 °C and boiled in 1x SDS loading buffer in the presence or absence of 100 mM DTT for 5 min at 95 °C.
4
Synthesis and Purification of Radioactive DNA Oligonucleotides
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All DNA oligonucleotides were synthesized
by the Integrated DNA Technologies (IDT; Coralville, IA). The radionucleotide
[γ-32P]ATP (6000 mCi/mmol) was purchased from MP
Biomedicals (Santa Ana, CA). T4 polynucleotide kinase and deoxynucleoside
5′-triphosphates were purchased from Thermo Scientific (Pittsburgh,
PA). Micro Bio-Spin TM 6 Columns were purchased from Bio-Rad (Hercules,
CA). All other chemicals were purchased from Thermo Scientific (Pittsburgh,
PA) and Sigma-Aldrich (St. Louis, MO). Purified human DNA polymerase
β (pol β) was purified following the procedures described
previously.54 (link),55 (link) The Klenow fragment was obtained
from New England Biolabs (Ipswitch, MA).
by the Integrated DNA Technologies (IDT; Coralville, IA). The radionucleotide
[γ-32P]ATP (6000 mCi/mmol) was purchased from MP
Biomedicals (Santa Ana, CA). T4 polynucleotide kinase and deoxynucleoside
5′-triphosphates were purchased from Thermo Scientific (Pittsburgh,
PA). Micro Bio-Spin TM 6 Columns were purchased from Bio-Rad (Hercules,
CA). All other chemicals were purchased from Thermo Scientific (Pittsburgh,
PA) and Sigma-Aldrich (St. Louis, MO). Purified human DNA polymerase
β (pol β) was purified following the procedures described
previously.54 (link),55 (link) The Klenow fragment was obtained
from New England Biolabs (Ipswitch, MA).
5
Recombinant SaGAPDH Activity Assay
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Recombinant SaGAPDH activity was determined by measuring the absorbance change at 340 nm and 25°C resulting from the production of NADH. The reaction was carried out in a 150 µl assay mixture containing 20 mM Tris–HCl (pH 8.7), 0.36 µM SaGAPDH, 1.25 mM NAD+, 1.25 mM ethylenediaminetetraacetic acid and 15 mM sodium arsenate. The reaction was started by the addition of 0.25 mM glyceraldehyde 3-phosphate. Initial reaction rates were calculated as described recently [25 (link)], by determining the slope in the linear part of the curve during the first 80 s of the reaction (GraphPad, linear regression function). The percentage of SaGAPDH activity was calculated as: Rate of inactivated/Rate of untreated × 100%. The results are presented as mean ± SEM from at least three separate experiments.
For the inactivation experiments, SaGAPDH was preincubated with 1 µM, 10 µM, 100 µM and 10 mM H2O2 for 10 min or with 10 mM CoASSCoA for 30 min. About 2 µl of the mixture was then added to the assay mixture and the remaining activity was measured as described. To reduce it, the enzyme was incubated with 10 mM DTT for 15 min. After treatments, excess H2O2, CoASSCoA and DTT were removed using Micro Biospin 6 columns (Bio-Rad).
For the inactivation experiments, SaGAPDH was preincubated with 1 µM, 10 µM, 100 µM and 10 mM H2O2 for 10 min or with 10 mM CoASSCoA for 30 min. About 2 µl of the mixture was then added to the assay mixture and the remaining activity was measured as described. To reduce it, the enzyme was incubated with 10 mM DTT for 15 min. After treatments, excess H2O2, CoASSCoA and DTT were removed using Micro Biospin 6 columns (Bio-Rad).
6
Native Mass Spectrometry of Proteins
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Native mass spectrometry was carried out using the Exactive Plus EMR instrument (Thermo Scientific, San Jose, CA, Lu et al., 2015) that was externally calibrated using a 5 mg/mL CsI solution prepared in water. Prior to analysis, the protein samples were buffer-exchanged into 150 mM ammonium acetate, pH 7.5 using MicroBiospin-6 columns (Bio-Rad, Hercules, CA) that had been pre-equilibrated in the same buffer. Protein samples were introduced into the mass spectrometer using offline Au/Pd-coated borosilicate emitters (NanoES Spray Capillaries, Medium, ES380, Thermo Scientific) at a flow rate of 10–40 nL/min. Spectra were acquired over the range m/z 500 – 20,000 in positive ion mode, were averaged, and then exported for deconvolution and subsequent generation of the zero-charge mass values using PeakSeeker and Unidec36 (link),37 (link). Samples were analyzed with the following experimental parameters: spray voltage (0.8 – 1.5 kV), injection flatapole = 5; inter flatapole lens = 5; bent flatapole = 5; transfer multipole = 2; C-trap entrance lens = 2, source DC offset (25 V), fragmentation energies (CE = 25 and CID = 65), injection times (200 μsec), trapping gas pressure (7.5), resolution (17,500 arbitrary units), capillary temperature (250 °C), S-len RF levels (200 V), microscans (10), and AGC (1e6).
7
Radiolabeled Porphyrin Nanoparticles Synthesis
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Metal ions, such as Cu2+ could be loaded via chelation during or after drug loading. The 64Cu2+ chelation was carried out through the addition of 64CuCl2 (Washington University, MO, USA) to the PVA-porphyrin aqueous solution under stirring for 2 hrs at room temperature 47 (link), 69 . The free metal ions in the PPNs solution were removed via column filtration using Micro Bio-Spin 6 columns (Bio-Rad, Hercules, CA, USA). Instant thin-layer chromatography (ITLC) was immediately performed to evaluate the radiochemical purity and yield after the incorporation of 64Cu into the PPNs.
8
Coupling of Recombinant Proteins to Fluorescent Beads
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Prior to coupling, the recombinant proteins was desalted by gel filtration using MicroBio-Spin 6 columns (Bio-Rad, California, USA) based on the manufacture’s protocol to exchange the buffer from sodium azide or imidazole to PBS. All the antigens were quantified using Pierce BCA protein Quantification Kit (Thermo Scientific, USA). Coupling of proteins to fluorescent microspheres was performed as described by Karanikola et al. [30 ]. MAP MC10007 beads (Luminex, USA) were coupled by gD protein of ILTV and MAP MC10015 beads (Luminex, USA) were coupled by N protein of IBV. A certain number of beads was transferred to one coupling reaction tube, followed by 100 μL bead wash buffer and suspended in 80 μL bead activation buffer. Then 10 μL of 50 mg/ml EDAC (Sigma-Aldrich) and 10 μL of 50 mg/ml S-NHS (Sigma-Aldrich, Germany) were added in the reaction buffer. The reaction tube was gently vortexed for 20 min at room temperature (RT). PBS (pH 7.4, 150 μL) was added twice and the recombinant protein was added. Incubation was carried out at RT for 2 h. The coupled beads were centrifuged and washed. Ultimately, the beads were stored at 4 °C in 150 μL storage buffer in the darkness.
9
Reversible Tau CoAlation and Dimerization
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Prior to the in vitro CoAlation assay of the 2N3R and 2N4R tau isoforms, the proteins were incubated for 30 min at room temperature with DTT (10 mM). Micro Biospin 6 columns (BioRad), equilibrated with 1X PBS, were used to remove excess of DTT. 2N3R and 2N4R tau isoforms (8 μM) were incubated (40 min, 25°C) with H2O2 (200 μM) in the presence or absence of CoA (70 μM). To stop the reaction, 5 mM N-ethyl maleimide (NEM) was added, and the samples were incubated for 10 min at 25°C. NEM is a thiol alkylating agent.
To study the reversibility of tau CoAlation or dimerization, two additional samples were prepared. Following oxidation and/or CoAlated of the 2N3R and 2N4R tau isoforms, 5 mM DTT (reducing agent) was added to each sample. The samples were then incubated for 10 min at 25°C. The reaction was stopped by the addition of NEM. Following alkylation with NEM, the samples were boiled in 1X non-reducing loading buffer and separated by SDS-PAGE.
To study the reversibility of tau CoAlation or dimerization, two additional samples were prepared. Following oxidation and/or CoAlated of the 2N3R and 2N4R tau isoforms, 5 mM DTT (reducing agent) was added to each sample. The samples were then incubated for 10 min at 25°C. The reaction was stopped by the addition of NEM. Following alkylation with NEM, the samples were boiled in 1X non-reducing loading buffer and separated by SDS-PAGE.
10
SnoopCatcher-Mediated Protein-DNA Conjugation
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