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Explain in detail the purification of IgG from human serum by anion exchange of Sephadex a-50 and isocratic elution.
Accepted Answer
Purification of IgG from Human Serum by Anion Exchange Chromatography using Sephadex A-50 and Isocratic Elution:
1. Sample Preparation:
Serum is collected and clarified by centrifugation to remove any cellular debris.
The serum is then diluted with a buffer solution (e.g., phosphate buffer) to adjust the pH and ionic strength.
2. Anion Exchange Chromatography:
Sephadex A-50: A gel filtration medium consisting of cross-linked dextran beads with diethylaminoethyl (DEAE) groups attached. The DEAE groups are positively charged, allowing them to bind to negatively charged molecules like IgG.
Equilibration: The column is first equilibrated with the same buffer used for sample dilution to ensure optimal conditions for protein binding.
Loading: The diluted serum sample is loaded onto the column. IgG, being negatively charged at the chosen pH, binds to the DEAE groups on the Sephadex A-50.
Washing: The column is washed with the equilibration buffer to remove unbound proteins and other impurities.
3. Isocratic Elution:
High-salt Buffer: A buffer solution with a high salt concentration (e.g., NaCl) is used to disrupt the ionic interactions between IgG and the DEAE groups on the resin. The salt ions compete with IgG for binding sites, effectively eluting the IgG from the column.
Constant Flow Rate: The high-salt buffer is passed through the column at a constant flow rate to maintain consistent elution conditions.
Fraction Collection: The eluate is collected in fractions, with the IgG-containing fractions identified based on protein concentration or specific activity.
4. Purification Assessment:
Protein Concentration: Determine the concentration of IgG in the collected fractions using methods like Bradford assay or UV-Vis spectrophotometry.
SDS-PAGE Analysis: Analyze the purity of IgG in the collected fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess the presence of any contaminating proteins.
Note: The pH of the buffer and the salt concentration used for elution can be adjusted depending on the specific properties of the IgG and the desired purity level.
1. Sample Preparation:
Serum is collected and clarified by centrifugation to remove any cellular debris.
The serum is then diluted with a buffer solution (e.g., phosphate buffer) to adjust the pH and ionic strength.
2. Anion Exchange Chromatography:
Sephadex A-50: A gel filtration medium consisting of cross-linked dextran beads with diethylaminoethyl (DEAE) groups attached. The DEAE groups are positively charged, allowing them to bind to negatively charged molecules like IgG.
Equilibration: The column is first equilibrated with the same buffer used for sample dilution to ensure optimal conditions for protein binding.
Loading: The diluted serum sample is loaded onto the column. IgG, being negatively charged at the chosen pH, binds to the DEAE groups on the Sephadex A-50.
Washing: The column is washed with the equilibration buffer to remove unbound proteins and other impurities.
3. Isocratic Elution:
High-salt Buffer: A buffer solution with a high salt concentration (e.g., NaCl) is used to disrupt the ionic interactions between IgG and the DEAE groups on the resin. The salt ions compete with IgG for binding sites, effectively eluting the IgG from the column.
Constant Flow Rate: The high-salt buffer is passed through the column at a constant flow rate to maintain consistent elution conditions.
Fraction Collection: The eluate is collected in fractions, with the IgG-containing fractions identified based on protein concentration or specific activity.
4. Purification Assessment:
Protein Concentration: Determine the concentration of IgG in the collected fractions using methods like Bradford assay or UV-Vis spectrophotometry.
SDS-PAGE Analysis: Analyze the purity of IgG in the collected fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess the presence of any contaminating proteins.
Note: The pH of the buffer and the salt concentration used for elution can be adjusted depending on the specific properties of the IgG and the desired purity level.
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