2 mg of purified wild-type or point-mutant SLX4BTB (20 mg/ml) was loaded onto a calibrated Superdex 75 column (volume 120 ml; GE Healthcare) equilibrated in 25 mM pH 8.0 Tris-HCl, 150 mM NaCl and 2 mM DTT. The column was eluted at a flow rate of 1 ml/min, and 1 ml fractions were collected, analyzed by SDS–PAGE and stained with Coomassie brilliant blue. Apparent molecular masses were calculated from the corresponding elution volumes using a calibration curve that was obtained with gel filtration standard proteins (Bio-Rad).
Gel filtration standard proteins
Manufactured by Bio-Rad
Sourced in United States
Gel filtration standard proteins are a set of proteins with known molecular weights that are used to calibrate and evaluate the performance of gel filtration chromatography systems. These proteins serve as molecular weight markers, allowing the estimation of the molecular weights of unknown samples separated by gel filtration.
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Lab products found in correlation
2 protocols using gel filtration standard proteins
1
Purification and Size Characterization of SLX4 Protein
2
Size-Exclusion Chromatography for Molecular Mass
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Size-exclusion chromatography was performed using a Superose 6 10/300 GL analytical column (GE Healthcare, Piscataway, NJ, USA) connected to an ÄKTAxpress chromatography system (GE Healthcare, Piscataway, NJ, USA) at 4°C. The column was equilibrated with 50 mM Tris-Cl and 150 mM NaCl at a pH of 7.5. The purified protein was loaded onto the analytical column at a concentration of 10 mg/mL and eluted at a flow rate of 0.3 ml/min. The native molecular mass of 12β-HSDH was determined by comparing its elution volume to that of Gel Filtration Standard proteins (Bio-Rad, Hercules, CA, USA): thyroglobulin, γ-globulin, ovalbumin, myoglobin, vitamin B12.
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