Labeling of recombinant probes was performed by conjugation of Meso Scale Discovery (MSD) Gold sulfo-TAG NHS-Ester and biotin to primary amines, as previously described [23 (link)]. Briefly, recombinant HIP protein was mixed with sulfo-TAG, incubated at RT for 2 h, and purified on a desalting spin column (Thermo Scientific, Waltham, MA, USA). Labeling efficiency was determined by spectrophotometry (450 nm), and protein concentration by a bicinchoninic acid (BCA) kit (Sigma Aldrich, St. Louis, MO, USA). Biotin labeling of PI HIP recombinant protein was performed using a biotinylation kit (Thermo Scientific), and products purified with a desalting spin column. Protein concentration and labeling efficiency were determined by spectrophotometry (500 nm) and a BCA kit, respectively.
Spin desalting column
Manufactured by Thermo Fisher Scientific
Sourced in United States
Spin desalting columns are a type of lab equipment used to remove small molecules, such as salts, from protein solutions. The columns contain a porous resin that allows small molecules to pass through while retaining larger proteins. This process, known as gel filtration chromatography, allows for the efficient separation and purification of proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Galectin 3, by Merck Group (2 mentions)
Mertk, by Sino Biological (2 mentions)
Pherastar plus, by BMG Labtech (2 mentions)
Trypsin, by Promega (2 mentions)
Irdye800cw n hydroxysuccinimide ester, by LI COR (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Plv mcherry, by Addgene (2 mentions)
Dulbecco s modified eagle medium, by American Type Culture Collection (2 mentions)
Phoenix cells, by American Type Culture Collection (2 mentions)
Human dermal microvascular endothelial cells hdmvecs, by ScienCell (2 mentions)
Endothelial cell culture medium, by ScienCell (2 mentions)
C57bl 6j wild type and ldl receptor ldlr ko b6.129s7 ldlrtm1her j mice, by Jackson ImmunoResearch (2 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Specord 50, by Analytik Jena (1 mentions)
Lys c, by Fujifilm (1 mentions)
Cetuximab, by LI COR (1 mentions)
Control igg, by Innovative Research (1 mentions)
Mini extruder, by Avanti Polar Lipids (1 mentions)
32 protocols using spin desalting column
1
Labeling of Recombinant Proteins
2
Protein Digestion and Peptide Purification
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Digestion of protein (200 μg for each sample) was performed according to the FASP procedure described by Wisniewski et al. [21 (link)]. Take 200 μg of each sample for enzymatic hydrolysis; the steps are as follows: for each sample, add DTT to 100 mM, boil for 5 min, and cool to room temperature. Add 200 μL UA buffer (8M Urea, 150 mM Tris-HCl, pH 8.0) and mix well. Transfer to 10 KD ultrafiltration centrifuge tube and centrifuge at 12,000× g for 15 min. Repeat the operation once and discard the filtrate. Add 100 μL iodoacetamide (IAA) (50 mM IAA in UA), cool to room temperature, and centrifuge. Then, add 100 μL UA buffer and centrifuge, and repeat 2 times. Add 100 μL NH4HCO3 buffer (50 mM) and centrifuge; repeat 2 times. Then, add 60 μL Trypsin buffer (6 μg Trypsin in 40 μL NH4HCO3 buffer), shake, and let it stand at 37 °C for 16–18 h. Replace with a new collection tube, centrifuge, collect the filtrate, add 0.1% trifluoroacetic acid (TFA) solution to redissolve it, and then desalt it using a Thermo desalting spin column. The filtrate was desalted using a Thermo desalting spin column, and the peptides were quantified.
3
Galectin-3 Binding Kinetics Assay
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Galectin-3 (Sigma-Aldrich) was labeled with Alexa Fluor 488 NHS Ester (succinimidyl ester) in PBS at room temperature for 30 min, and free dye was washed with desalting spin columns (Thermo Scientific). Labeling was checked by MALDI mass spectrometry. Fluorescence anisotropy measurements were recorded using a PHERAstar Plus multidetection plate reader (BMG Labtech) equipped with a fluorescence polarization optic module (λex = 485 nm, λem = 520 nm) at 25°C. Each data point is the mean of 200 flashes per well. The voltage gain was set by adjusting the target mP values of labeled Gal-3 to that of fluorescein (35 mP). Serial dilutions of MerTK (Sino Biological), modified citrus pectin (ecoNugenics), and TREM2 ectodomain were made in PBS and added together in a 1:1 ratio to a constant concentration of Gal-3 (35 nM; 44 mP). Kd was estimated, assuming a 1:1 binding, using GraphPad Prism. Purified TREM2 ectodomain was a kind gift from Roger Dodd and Peter St. George-Hyslop (University of Cambridge).
4
Characterizing hsp60-hsp10 Chaperone Complexes
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All experiments were performed on a Malvern Zetasizer Nano ZS instrument. For the ATP-bound conformation, 1 µM hsp60 (wild type or mutant) was added to 2 µM hsp10, 100 mM ATP, and 100 mM MgCl2. For the ADP-bound conformation, 1 µM hsp60 (wild type or mutant) was added to 2 µM hsp10, 2 mM ADP. The samples were incubated for 5 minutes at 37 °C upon addition of nucleotides. The excess unbound nucleotides were removed with desalting spin columns (Thermo Scientific). All the data are reported as percent volume to normalize the measurement with the amount of protein at each size. 180 measurements were averaged together to give a statistically significant size distribution that describes the hydrodynamic diameter of the complexes.
5
Galectin-3 Binding Affinity Assay
6
Cyto-Oxidative Effects on Malate Dehydrogenase
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The different cyMDH samples (1 µg/µl) were pre-reduced in the presence of 0.5 mM DTT for 30 min at room temperature in exchange buffer (35 mM Tris–HCl, pH 8.8). Before the oxidative treatment was started, enzyme activities of reduced cyMDHs were recorded. To determine the cyMDH activity, proteins were diluted to a final concentration of 2 ng/µl in reaction buffer (0.1 M Tris–HCl, pH 8.0, bovine serum albumin (0.1 mg/ml), 10 mM MgCl2, 0.2 mM NADH). Cuvettes were filled with 985 µl of reaction buffer and 5 µl sample containing 10 ng protein. After baseline determination, the substrate oxaloacetate (OAA) (1 mM) was added and the cyMDH activity recorded at 334 nm for 5 min using a spectrophotometer (SPECORD 50, Analytik Jena). After activity determination of the fully reduced enzyme, the DTT-containing samples were desalted (Desalting Spin Columns, Thermo Scientific). After the removal of DTT, the proteins were oxidized by incubation with 0.5 mM diamide for 30 min. To estimate the protective properties of small molecules on cyMDH oxidation, GSH, NAD+, NADH, NADP+, NADPH, OAA, or malate, at the given concentrations, were added before the oxidative treatment. After determining the activity of the oxidatively treated cyMDHs, 20 mM DTT was used to check the reversibility of the oxidation process. Data are means from three independent experiments with three technical replicates each.
7
Proteomic Quantification of Drug Responses
8
Labeling Antibodies with IRDye800CW
9
Cetuximab-IRDye800CW Conjugation Protocol
10
Labeling Antibodies with IRDye800CW
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