Assembly sizes of sHsps (1 µM) were determined by size exclusion chromatography (S200; GE Healthcare) in buffer E (50 mM Tris pH7.5, 20 mM NaCl, 5% glycerol [v/v], 2 mM DTT, pH 7.5). Molecular weights and oligomeric states of sHsps were calculated from a calibration curve generated from a size exclusion standard (Biorad).
Size exclusion standard
Manufactured by Bio-Rad
Sourced in Poland
Size exclusion standard is a laboratory tool used to determine the molecular weight distribution of macromolecules in a sample. It functions by separating molecules based on their size as they pass through a porous gel matrix, allowing for the accurate measurement of their molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium acetate, by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Hydrogen peroxide, by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Copper sulfate, by Merck Group (2 mentions)
Nitric acid, by Merck Group (2 mentions)
Zinc acetate, by Merck Group (2 mentions)
Glycine, by Merck Group (2 mentions)
Rutin, by Merck Group (2 mentions)
Zinc sulfate, by Merck Group (2 mentions)
Superose 12 column, by GE Healthcare (2 mentions)
Sodium hydrogen carbonate, by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Pepsin from porcine gastric mucosa, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Tris hydroxymethyl aminomethane, by Merck Group (1 mentions)
Superdex 200 10 300 size exclusion column, by GE Healthcare (1 mentions)
Superose 6 10 300 size exclusion column, by GE Healthcare (1 mentions)
10 protocols using size exclusion standard
1
Size Exclusion Analysis of sHsps
2
Proteomic Analysis of Gastric Mucosa
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Pepsin from porcine gastric mucosa and pancreatin were of biological grade (Sigma-Aldrich, Buchs, Switzerland). Sodium chloride, ammonium acetate, sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane, hydrogen peroxide, sodium hydrogen carbonate, and hydrochloric acid were also purchased from Sigma-Aldrich and were of analytical reagent grade. Nitric acid was the product of Fluka (Switzerland) of purity for trace metals analysis. Formic acid of LC/MS purity was obtained from Fisher Scientific (Fair Lawn, NJ, USA). Methanol (LC–MS grade) was purchased from POCH (Gliwice, Poland). Deionized water (18 MΩ cm) prepared with a Milli-Q system (Millipore Elix 3, Millipore, Saint-Quentin, France) was used throughout. The SEC column was calibrated using size exclusion standard (BIO-RAD, Warsaw, Poland).
3
Quantification of Trace Elements in Plant-Based Drinks
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The plant-based drinks and raw materials: oat, rice, almond, soybean, millet, and coconut-rice, were purchased from different Polish markets. A certificated reference material (IRMM-804 rice flour, Belgium) was analyzed to validate the results of the total arsenic concentrations.
The reagents used in analysis were of analytical reagent grade purchased from Sigma-Aldrich (Sigma-Aldrich, Buchs, Switzerland). The nitric acid of purity for trace metal analysis was purchased from Fluka (Buchs, Switzerland). Ultra-pure water (15 MΩ cm) was obtained with Milli-Q Elix 3 Water Purification system Millipore (Molsheim, France).
The SEC column was calibrated, using the size exclusion standard (BIO-RAD, Warsaw, Poland). The calibration curves were prepared using a solution of Environmental Spike Mix (1000 mg L−1 of Fe, K, Ca, Na, Mg and 100 mg L−1 of Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Tl, V, Zn, U; matrix 5% HNO3) purchased from Agilent Technologies.
The reagents used in analysis were of analytical reagent grade purchased from Sigma-Aldrich (Sigma-Aldrich, Buchs, Switzerland). The nitric acid of purity for trace metal analysis was purchased from Fluka (Buchs, Switzerland). Ultra-pure water (15 MΩ cm) was obtained with Milli-Q Elix 3 Water Purification system Millipore (Molsheim, France).
The SEC column was calibrated, using the size exclusion standard (BIO-RAD, Warsaw, Poland). The calibration curves were prepared using a solution of Environmental Spike Mix (1000 mg L−1 of Fe, K, Ca, Na, Mg and 100 mg L−1 of Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Tl, V, Zn, U; matrix 5% HNO3) purchased from Agilent Technologies.
4
Analytical Reagents for Biochemical Assays
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Sodium chloride, ammonium acetate, rutin, glycine, nitric acids, hydrogen peroxide, zinc acetate, zinc sulfate, copper sulfate, and hydrochloric acid were purchased from Sigma Aldrich with analytical reagent grade. Pepsin from porcine gastric mucosa and pancreatin was of biological grade (Sigma-Aldrich, Buchs, Switzerland). Deionized water (18 MΩ cm) prepared with a Milli-Q system (Millipore Elix 3, Millipore, Saint-Quentin, France) was used throughout. The SEC column was calibrated using the size exclusion standard (BIO-RAD, Warsaw, Poland).
5
Analytical Reagents for Biochemical Assays
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Sodium chloride, ammonium acetate, rutin, glycine, nitric acids, hydrogen peroxide, zinc acetate, zinc sulfate, copper sulfate, and hydrochloric acid were purchased from Sigma Aldrich with analytical reagent grade. Pepsin from porcine gastric mucosa and pancreatin was of biological grade (Sigma-Aldrich, Buchs, Switzerland). Deionized water (18 MΩ cm) prepared with a Milli-Q system (Millipore Elix 3, Millipore, Saint-Quentin, France) was used throughout. The SEC column was calibrated using the size exclusion standard (BIO-RAD, Warsaw, Poland).
6
Size Exclusion Chromatography Protocol
7
Structural Impact of NudT16 Mutations
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An additional step of size exclusion was performed using a Superdex 75 10/300 to determine whether the quaternary structure of HsNudT16 is affected by H24W, F36A, F61S, F36A F61S or Δ17 mutation. 100 µL of each purified HsNudT16 mutant was loaded onto the column at a rate of 0.3 mL/min and was eluted over one column volume. Size exclusion standards from Bio-Rad (catalog number: 151–1901) were run using the same method.
8
Protein Complex Size Estimation
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For size estimations, 500 μL of the eluted protein was loaded onto Superdex 200 10/300 size exclusion column (GE Healthcare) or Superose 6 10/300 size exclusion column (GE Healthcare). Fractions of 500 μL were collected from 7 or 8 mL to 24 mL. Fractions were analyzed by SDS-PAGE to identify factions containing the protein of interest and with EMSAs to determine DNA-binding activity. Size exclusion standards (Bio-Rad) ranging from 1.35 to 670 kDa were used to calculate the partition coefficients and estimate the sizes of the protein complexes.
9
Chromatographic Analysis of Cpl-1 Mutants
10
Cpl-1 Mutant Analysis via SEC
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Cpl-1 and its mutants were subjected to analytical size exclusion chromatography on a Superose 12 column (GE Healthcare) as previously described. 16 The column was calibrated with size exclusion standards (Bio-Rad) and equilibrated with either PBS or PBS supplemented with choline chloride at indicated concentrations for two column volumes (CV) before loading protein sample. 500 μL of a 1.5 mg/mL protein sample was loaded onto the column for each run, and the sample was eluted for 1.5 CV. Alternatively, phosphocholine chloride calcium salt tetrahydrate was used in 10 mM Tris (pH 7.4) with 150 mM NaCl because it was insoluble in PBS.
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