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Gel Filtration Chromatography

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Textbook on Cloning, Expression and Purification of Recombinant Proteins

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Abstract

Apart from finding multitudes of applications in chemical, medicinal, and pharmaceutical research, gel filtration chromatography (GFC) has also become a routine tool in almost every biomedical research laboratory especially protein biochemistry. With rapid advancement and implementation of recombinant DNA technology in basic research, the requirement of purifying bacterially expressed proteins for further characterization has increased manifold. Gel filtration chromatography is usually adopted in the last or polishing purification step to obtain highly purified proteins that are later used for biophysical and structural studies. The versatility and robustness of GFC lies in the fact that the protein molecules do not adhere to the column during separation unlike ion-exchange and affinity chromatography. This gives GFC a significant advantage as it allows proteins to be eluted in a buffer condition that is conducive toward their future applications or storage. Since GFC separates analytes as a function of their size or molecular weight, it is also used as an analytical tool to determine molecular weights and oligomeric properties of macromolecules. This chapter, which is a sequel to the previous two chapters on chromatographic techniques, describes the theory, instrumentation, and applications of gel filtration chromatography along with elaborate discussions on several important protocols and troubleshooting tips.

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Acknowledgments

The authors thank Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) for providing necessary infrastructure and resources for successful completion of the chapter. The authors acknowledge Ms. Chanda Baisane, Bose Lab, ACTREC for formatting the manuscript.

Author information

Authors and Affiliations

  1. Integrated Biophysics and Structural Biology Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Mumbai, India

    Raghupathi Kummari & Kakoli Bose

  2. Homi Bhabha National Institute, BARC Training School Complex, Mumbai, India

    Raghupathi Kummari & Kakoli Bose

Authors
  1. Raghupathi Kummari
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  2. Kakoli Bose
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Corresponding author

Correspondence to Kakoli Bose .

Editor information

Editors and Affiliations

  1. Integrated Biophysics and Structural Biology Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Mumbai, India

    Kakoli Bose

Problems

Problems

Multiple Choice Question

  1. 1.

    In Gel Filtration Chromatography the best resolution is usually achieved with the following combination:

    1. (a)

      Faster flow rate, short and wide columns, small pore-size gel, and less sample volumes (1–5% of the total column volume).

    2. (b)

      An optimum flow rate (slow or medium), long and narrow columns, small pore-size gel and sample volumes (1–5% of the total column volume).

    3. (c)

      Faster flow rate, short and wide columns, large pore-size gel, and less sample volumes (1–5% of the total column volume).

    4. (d)

      An optimum flow rate (slow or medium), long and narrow columns, large pore-size gel, and high sample volumes (10–25% of the total column volume).

  2. 2.

    Which one of the following statements is true?

    1. (a)

      Kav value of the protein of interest should fall between 0 and 1 and can be obtained empirically by substituting the values (known total, void, and elution volumes) in the standard equation.

    2. (b)

      Kav value of the protein of interest should fall between 0 and 1 and can be obtained experimentally from the standard graph (known molecular weight markers).

    3. (c)

      Kav value of the protein of interest should be any integer and can be obtained experimentally from the standard graph (known molecular weight markers).

    4. (d)

      Kav value of the protein of interest should be any integer and can be obtained empirically by substituting the values (known total, void, and elution volume) in the standard equation.

  3. 3.

    A gel filtration column is filled with Polyacrylamide media (Bio-Gel P-150) with a molecular weight separation range of 15–150 kDa. A mixture of four different proteins: Protein A (MW 55 kDa); Protein B (MW 35 kDa); Protein C has a molecular weight (MW 200 kDa), and Protein D (MW 10 kDa). Protein B has a tendency to form aggregates (>300 kDa) in small proportions. Therefore, the order of their elution time would be:

    1. (a)

      Protein D will be eluted first, followed by B, A, C and aggregates will be eluted last.

    2. (b)

      Aggregates will be eluted first, Protein C will be the second to elute, followed by A, B, and D.

    3. (c)

      Aggregates and Protein C will elute simultaneously in the void volume, followed by D, B, and finally A.

    4. (d)

      Aggregates and Protein C elute simultaneously in the void volume, followed by A, B, and finally D.

Subjective Question

  1. 1.

    A researcher was trying to characterize the native oligomeric status of one of the least studied member “PROTEIN X” of a member of a serine protease family. “PROTEIN X” has been purified using Ni-NTA chromatography and subsequently subjected to gel filtration chromatography using Superdex 200 10/300 HR column (Vt = 120 mL) (GE Healthcare, Uppsala, Sweden). The elution volumes with other necessary information are provided in the table below. Blue Dextran was used to determine the void volume of the column. The standard proteins BSA, MBP, and lysozyme were run on the same column and their elution volumes are provided. Answer the following two questions from the following information:

    1. (a)

      Calculate the Kav of the standards.

    2. (b)

      Find the Kav values and thereafter calculate the molecular weights and oligomerization status of newly characterized “PROTEIN X.”

      S. no

      Protein

      Mol. wt.

      Elution volume

      1

      Blue Dextran

      2000 kDa

      46.5 mL

      2

      BSA

      66.5 kDa

      77 mL

      3

      MBP

      45 kDa

      86.5 mL

      4

      Lysozyme

      14 kDa

      111.5 mL

      5

      “PROTEIN X”

      ?

      Peak 1: 64 mL (major peak)

      Peak 2: 87 mL (minor peak)

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Kummari, R., Bose, K. (2022). Gel Filtration Chromatography. In: Bose, K. (eds) Textbook on Cloning, Expression and Purification of Recombinant Proteins. Springer, Singapore. https://doi.org/10.1007/978-981-16-4987-5_8

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