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Methods:Affinity Chromatography

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Contents

Purpose

To separate molecules with a specific affinity from the rest of the solution.

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Materials Needed

  • Chromatography column, with matrix
  • ligand with desired specificity that can be coupled to the matrix
  • appropriate selection of buffer (at least running, washing and elution buffers)
  • fraction collector
  • activity assay

Protocol

Column Prep

  • pack column with matrix beads - What matrices are available? When would you use them?
    • some commercially available being:Thermo
    • Glutathione and nickel agarose are frequently used for purification of recombinant proteins containing GST and 6-Histidine tags, respectively.
    • make sure the pore size of the beads is appropriate for your protein, Molecular Kitchen, Wikikpedia - ?? How is this decided? What else matters here?
  • load ligand onto the column, this is not always necessary for some commercially available pre-prepared columns
    • Wash the column with multiple column volumes of running buffer to remove excess ligand
  • run the column using only a running buffer that will ensure the ligand remains bound properly
    • As the buffer passes through check the flowthrough for the presence of you ligand
  • leave a small volume sitting on top of the column to prevent it from drying out
    • If the Column is not kept under solution and hydrated, it will dry out and crack
      • time must then be spent either rehydrating the column in an effort to save it or completely repacked

Running the column

Loading

  • Load sample solution to be separated onto the column
    • depending on the set up this might be manual pouring onto column or injecting into a sample tube
      • make sure the column is fully hydrated before doing this
    • Use multiple (2-3) column volumes of running buffer to load the column
      • Collect all fractions that come off for analysis as a control

Washing

  • Switch from running buffer to washing buffer
    • the washing buffer should wash all unwanted molecules from the column, leaving only the protein(s) that interacted with the attached ligand
    • Use multiple (~5+) column volumes (if coupled to UV-Vis wait for the absorbance to return to baseline) of washing buffer to ensure the column is cleared
      • Collect all fractions that come off for analysis as a control

Eluting

  • Switch from washing buffer to elution buffer
    • the elution buffer should be specific for removing the interaction partners of the attached ligand
      • everything else should have already been removed by the washing buffer
    • Use multiple (2-3) column volumes of elution buffer to ensure everything has eluted
      • Collect all fractions that come off for analysis as they should have your desired sample

Fraction Collection

  • Collected fractions should be screened to determine which fractions to collect and keep for further use
  • Screening:
    1. Gel profiling using Gel Electrophoresis
    2. If the protein has an enzymatic activity then an activity assay can also be used.
    3. Bradford Assay can be used to approximate protein concentration
    4. Concentration can be estimated by measuring absorbance at A280 - see Pace et al. (1995) [1]. ProtParam will automatically calculate extinction coefficients for proteins of known sequence using the formula in Pace et al.
  • Fractions with the highest purity and/or activity can be pooled

Storage, Reuse & Recharging

Links

Amersham Biosciences

Kimball Biology

Bio-Medicine

Wikipedia

Jena Bioscience

Sigma-Aldrich

GE Healthcare

Pall

User notes

References

[2] [3] See Help:References for how to manage references in EcoliWiki.

  1. Pace, CN et al. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4 2411-23 PubMed EcoliWiki page
  2. Cuatrecasas, P et al. (1968) Selective enzyme purification by affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 61 636-43 PubMed EcoliWiki page
  3. Adrados, BP et al. (2001) Size exclusion behavior of hydroxypropylcellulose beads with temperature-dependent porosity. J Chromatogr A 930 73-8 PubMed EcoliWiki page