PD MiniTrap G10 columns (GE Healthcare, Solingen, Germany) were equilibrated with 6 column volumes of milli-Q water. Labeled samples, brought to a volume of 100 µL using Milli-Q water, were loaded onto the columns, which were subsequently washed with 550 µL milli-Q water. Samples were eluted with 1250 µL milli-Q water and dried under vacuum.
Pd minitrap g 10 column
Manufactured by GE Healthcare
Sourced in United Kingdom, Germany
The PD MiniTrap G-10 columns are desalting and buffer exchange columns designed for fast and efficient sample preparation. The columns are pre-packed with Sephadex G-10 resin, which effectively removes salts, buffers, and other low-molecular-weight compounds from protein, peptide, or nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Zetasizer nano s, by Malvern Panalytical (2 mentions)
Sephadex g 10 resin, by GE Healthcare (1 mentions)
Maltohexaose, by Biosynth (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Hplc grade water, by Thermo Fisher Scientific (1 mentions)
Speedvac concentrator, by Thermo Fisher Scientific (1 mentions)
Iodomethane, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
E coli bl21 de3 plyss, by Promega (1 mentions)
96 well half area plates, by Greiner (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Escherichia coli bl21 de3 plyss, by Promega (1 mentions)
Ppiczc, by Thermo Fisher Scientific (1 mentions)
6520 lc ms system, by Agilent Technologies (1 mentions)
Hplc grade methanol meoh, by Merck Group (1 mentions)
Water h2o, by Merck Group (1 mentions)
Disuccinimidyl suberate dss, by Merck Group (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
22 protocols using pd minitrap g 10 column
1
Protein Purification Using PD MiniTrap G10
2
Amyloid-beta Peptide Purification
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300 μL of AβM42 at 25.5
μM was buffer exchanged into 100 mM Na2HPO4 pH 7 using PD MiniTrap G-10 columns (GE Healthcare) following the
gravity exchange protocol provided. The buffers were kept ice-cold
to reduce oligomerization. 500 μL of the same aliquot of AβM42
at 25.5 μM was also injected into a Superdex 75 10/300 GL gel
filtration column. The 100 mM Na2HPO4 pH 7 buffer
was kept on ice and the column wrapped in an ice bag to keep the protein
as cold as possible. The AβM42 was eluted isocratically at 0.8
mL/min and eluted between 17.5 and 18 min, as expected for a protein
of 4.5 kDa.
μM was buffer exchanged into 100 mM Na2HPO4 pH 7 using PD MiniTrap G-10 columns (GE Healthcare) following the
gravity exchange protocol provided. The buffers were kept ice-cold
to reduce oligomerization. 500 μL of the same aliquot of AβM42
at 25.5 μM was also injected into a Superdex 75 10/300 GL gel
filtration column. The 100 mM Na2HPO4 pH 7 buffer
was kept on ice and the column wrapped in an ice bag to keep the protein
as cold as possible. The AβM42 was eluted isocratically at 0.8
mL/min and eluted between 17.5 and 18 min, as expected for a protein
of 4.5 kDa.
3
Fluorescent 2-AA Glycan Labeling
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To enhance the detection signal of the N‐glycans, reduce the heterogeneity of signal strength from different glycans, and enable separation by C18 chromatography, we derivatized the glycans with 2‐AA prior to analysis by nano‐LC–MS (Fig. 4 ). The labeling of N‐glycans with 2‐AA was performed by reductive amination using a protocol adapted from Sigma‐Aldrich. A labeling solution containing 60 mg·mL−1 2‐AA and 6 0 mg·mL−1 sodium cyanoborohydride in acetic acid : DMSO (30 : 70) was heated briefly in a 65 °C heating block followed by addition of H2O to a final concentration of 10%. Five microliters of this labeling solution was added to the lyophilized N‐glycans and incubated at 65 °C for 3 h. The reaction was stopped by the addition of 95 µL H2O, and the labeled N‐glycans were immediately purified and desalted by size exclusion chromatography using PD minitrap G‐10 columns (GE healthcare, Chicago, Ill, USA). The columns were equilibrated with 10 mL H2O prior to the application of sample in a volume of 100 µL. After washing with 850 µL H2O, labeled N‐glycans were eluted in 250 µL H2O. This fraction was stored at −20 °C until the sample was analyzed by nano‐LC/MS.
4
Radiolabeling of PSMA Ligand NG001 with Lead-212
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The PSMA ligand NG001 was supplied as HPLC purified and dried trifluoroacetic acid salt (purity of ≥98%) by MedKoo Biosciences (Morrisville, NC, USA). NG001 dissolved in 0.5 M ammonium acetate in 0.1 M HCl was labelled with 212Pb using a liquid 224Ra/212Pb generator solution, as previously described [25 (link),68 (link)]. The 212Pb-NG001 was purified using PD Minitrap G-10 columns prepacked with Sephadex G-10 resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) to remove free 224Ra to a level below 0.8%. The radiochemical purity (RCP) of the radioligand was measured by thin-layer chromatography, and radioligands with RCP >95% were used for the experiments.
5
Glycan Labeling and Methylation Protocol
6
Recombinant GST-LifeAct-GFP Expression
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Expression of GST-LifeAct-GFP was induced in E. coli BL21 (DE3-pLysS) (Promega) using 0.5 mM IPTG at 30°C for 4 hr. The recombinant protein was purified on Glutathione sepharose 4B beads according to the manufacture’s instructions (GE Healthcare). The elution buffer containing glutathione was exchanged to reactivation buffer (0.16 M sucrose, 5 mM MgCl2, 50 mM potassium acetate, 20 mM MOPS–NaOH [pH 7.0], pH adjusted to 7.5) using PD Minitrap G-10 columns (GE Healthcare). The purified recombinant proteins were stored at −80°C.
7
Quantifying Amyloid-beta Aggregation Kinetics
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20 μM
freshly made ThT (Abcam, Cambridge, UK) was added to
50 μL of 5 μM AβM42 after buffer exchange into 100
mM Tris, 100 mM NaCl pH 7 using PD MiniTrap G-10 columns (GE, Healthcare).
All samples were loaded onto nonbinding, clear-bottom, 96-well half
area plates (Greiner Bio-One GmbH, Germany). The plates were sealed
with a SILVERseal aluminum microplate sealer (Grenier Bio-One GmbH).
Fluorescence measurements were taken with a FLUOstar Omega plate reader
(BMG LABTECH GmbH, Ortenbery, Germany). The plates were incubated
at 37 °C with double orbital shaking at 300 rpm for 1 min before
each was read every 5 min for 600 min. Excitation was set at 440 nm
with 20 flashes and the ThT fluorescence intensity measured at 480
nm emission with a 1300 gain setting. Two ThT assays were run using
four fractions of AβM42 from two purification runs with four
wells per fraction. Data were normalized to the sample with the maximum
fluorescence intensity for each plate.
freshly made ThT (Abcam, Cambridge, UK) was added to
50 μL of 5 μM AβM42 after buffer exchange into 100
mM Tris, 100 mM NaCl pH 7 using PD MiniTrap G-10 columns (GE, Healthcare).
All samples were loaded onto nonbinding, clear-bottom, 96-well half
area plates (Greiner Bio-One GmbH, Germany). The plates were sealed
with a SILVERseal aluminum microplate sealer (Grenier Bio-One GmbH).
Fluorescence measurements were taken with a FLUOstar Omega plate reader
(BMG LABTECH GmbH, Ortenbery, Germany). The plates were incubated
at 37 °C with double orbital shaking at 300 rpm for 1 min before
each was read every 5 min for 600 min. Excitation was set at 440 nm
with 20 flashes and the ThT fluorescence intensity measured at 480
nm emission with a 1300 gain setting. Two ThT assays were run using
four fractions of AβM42 from two purification runs with four
wells per fraction. Data were normalized to the sample with the maximum
fluorescence intensity for each plate.
8
Purification of Recombinant Actin-Binding Proteins
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Recombinant GST-LifeAct-GFP was expressed in Escherichia coli BL21 (DE3-pLysS; Promega) using 0.5 mM IPTG for induction at 30°C for 4 h. The protein was purified on glutathione Sepharose 4B beads according to the manufacturer’s instructions (GE Healthcare). The elution buffer containing glutathione was exchanged with reactivation buffer (0.16 M sucrose, 5 mM MgCl2, 50 mM potassium acetate, and 20 mM MOPS-NaOH, pH 7.0; pH adjusted to 7.5) using PD Minitrap G-10 columns (GE Healthcare). The purified proteins were stored at −80°C. Recombinant GST-Cdc3 and GST-Cdc12(FH1FH2) were expressed in E. coli and purified. The GST-tag of GST-Cdc3 was cleaved off with Factor Xa protease, and the GST tag was removed by incubating with GSH-Sepharose. Proteins were dialyzed against G-buffer (5 mM Hepes, pH 7.4, 0.2 mM CaCl2, 0.01% [wt/vol] NaN3, and 0.5 mM DTT) without ATP and stored at −80°C. The human β-actin cDNA with His-Tag at the C terminus was cloned into pPICZc (Invitrogen) and expressed in Pichia pastoris. The recombinant protein was purified using a Ni-NTA column, the His-Tag was cleaved off using chymotrypsin, and the protein was stored in G-buffer containing 0.2 mM ATP.
9
Peptide Synthesis and Purification for Biophysical Studies
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Peptides (wild-type or modified) used in biophysical or crystallographic experiments were synthesized by the TUFTS Core Facility, on a 0.1 mmol scale with one round of HPLC purification. Peptides were re-suspended into water or buffer (50 mM HEPES pH 7.5, 150 mM NaCl) based on their overall charge. All peptide solutions were then further purified using PD MiniTrap G-10 columns (GE Healthcare Life sciences) according to the manufacturer’s instructions, to remove any remaining chemical residuals from the synthesis. Peptide details are summarized in Table S1 E.
10
Optimized N-glycan Purification Protocol
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This protocol has been adapted from the manufacturer's protocol. The washing and elution volumes of the protocol were optimized by measuring the fluorescence intensity of eluted fractions in a fluorescence plate reader (data not shown). The PD minitrap G‐10 columns (GE Healthcare 28‐9180‐10) were equilibrated with 10 ml H20 with gravity flow. The sample was added in 100 µl volume (5 µl sample + 95 µl H20), and the flow‐through collected. The column was washed with 850 µl H20, which was pooled with the first flow‐through. Labeled N‐glycans were eluted with 250 µl H20 and collected in a new marked vial. The sample was stored at −20°C in darkness until analyzed with LC‐MS.
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