Which type of chromatography would be used to separate and determine the RMM of two unknown bacterial proteins?
To separate and determine the relative molecular mass (RMM) of two unknown bacterial proteins, one could use gel permeation chromatography (GPC), also known as size exclusion chromatography (SEC).
Here's an explanation of how to use GPC for this purpose:
1. Sample Preparation: Extract the proteins from the bacterial cells using appropriate techniques, such as cell lysis and protein extraction. Ensure that the proteins are in a soluble and purified form before proceeding.
2. Column Selection: Choose a GPC column with a suitable pore size range that allows for the separation of protein samples based on their molecular weight. The column should have a stationary phase consisting of porous beads through which protein molecules of different sizes can freely pass.
3. Mobile Phase: Select a compatible mobile phase, typically a buffer solution that maintains protein stability and prevents protein aggregation. The mobile phase should be able to flow through the column efficiently.
4. Calibration Standards: Prepare a set of protein standards with known molecular weights that span the expected range of the unknown proteins. These standards will be used to correlate the elution volume with the molecular weight.
5. Column Packing: Properly pack the GPC column with the stationary phase, taking care to ensure a uniform and highly efficient packing.
6. Sample Injection: Load the protein mixture, including the unknown proteins, onto the GPC column. The proteins will migrate through the column based on their size or molecular weight.
7. Elution and Detection: As the mobile phase passes through the column, the proteins will elute at different rates, with larger proteins eluting faster than smaller ones. Measure the elution volumes of the unknown proteins and the calibration standards.
8. Molecular Weight Determination: Create a calibration curve by plotting the elution volumes of the protein standards against their known molecular weights. Use this curve to estimate the molecular weights of the unknown proteins based on their respective elution volumes.
By following these steps, gel permeation chromatography can be employed to separate and determine the RMM of two unknown bacterial proteins.