19.03.2023 - 20:52

You have a gel filtration column with an unknown matrix. Describe step-by-step how you would determine its void volume and exclusion limit.

Question:

You have a gel filtration column with an unknown matrix. Describe step-by-step how you would determine its void volume and exclusion limit.

Answers (1)
  • Gargantua
    April 11, 2023 в 06:02

    To determine the void volume and exclusion limit of a gel filtration column with an unknown matrix, the following step-by-step procedure can be followed:

    1. Prepare a set of standard molecular weight markers of known sizes. These should be selected so that they span a wide range of molecular weights.

    2. Prepare a sample containing a mixture of proteins of different sizes.

    3. Equilibrate the gel filtration column with an appropriate buffer to remove any storage buffer or other impurities that may interfere with the separation.

    4. Load a small amount of the sample onto the column and collect fractions as they elute from the column.

    5. Measure the absorbance of each fraction at a suitable wavelength to detect the presence of the proteins in the sample.

    6. Plot the elution profile of the molecular weight markers on a graph of elution volume versus molecular weight.

    7. Identify the elution volumes corresponding to the void volume and the exclusion limit of the column. The void volume is the volume of the column that is not occupied by the matrix and represents the largest molecules that can pass through the column without interacting with the matrix. The exclusion limit is the molecular weight of the smallest molecule that is excluded from the matrix and does not enter the pores of the column.

    8. Plot the elution profile of the sample on the same graph as the molecular weight markers.

    9. Determine the molecular weights of the proteins in the sample by comparing their elution volumes with those of the molecular weight markers.

    10. Verify that the molecular weights obtained for the sample are consistent with the void volume and exclusion limit of the column.

    By following this step-by-step procedure, it is possible to determine the void volume and exclusion limit of a gel filtration column with an unknown matrix and to use this information to analyze the molecular weight of proteins in a mixture.

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