Download Hydrophobic Interaction Chromatography (HIC

CityLab’s SummerLab
Hydrophobic* Interaction Chromatography (HIC)
protocol for the protein purification station
*Hydrophobic literally means “water fearing.” It refers to substances that do not
mix well with water (i.e., Oil is hydrophobic). Note how it stays together in small
beads and does not mix with water.
Purpose: To separate hydrophobic proteins based on their tendency to stick to
specially treated beads when the proteins are in a salty solution.
Outcome: Hydrophobic proteins that become sticky in salty solutions will be
separated from proteins that are not as sticky in salty solutions.
Receptor
surface
Hydrophobic
proteins
Adapted from BioRad Biotechnology Explorer® Green Fluorescent Protein
Purification Kit Catalog number 166-0005-EDU
1
CityLab’s SummerLab
2
I. Prepare the column
The addition of a medium salt solution (equilibration buffer) insures a consistent (even)
salt consistency to the beads so hydrophobic proteins will stick to them.
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Label tubes 1, 2, 3 etc.
Add 2 ml of equilibration buffer to the
column.
Remove the column cap and let the
equilibration buffer drain into collection tube
#1 until the buffer reaches the surface of the
HIC matrix.
Adapted from BioRad Biotechnology Explorer® Green Fluorescent Protein
Purification Kit Catalog number 166-0005-EDU
CityLab’s SummerLab
3
II. Add a salt solution to the Lysate sample
Addition of a high salt solution to hydrophobic proteins and makes the proteins stick to
the beads.
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Add 150 µl of Lysate to an empty microcentrifuge tube.
Add 150 µl of binding buffer to the Lysate sample. (Always add binding buffer
to the sample in a 1:1 ration. i.e. The sample volume is 150 µl, therefore add 150 µl
of binding buffer.)
III. Load the Column
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Add 300 µl of the Lysate sample with binding buffer
prepared in part II to the surface of the beads.
HINT: Hold the tip of the pipette against the side of the column
just above the beads. Gently pipette the sample so that it falls
gently onto the beads with minimal disruption.
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Drain the column into collection tube #1 until the buffer level is at the top of
the beads.
Notebook Entry: Did the GFP stick to the beads? Why or why not?
Adapted from BioRad Biotechnology Explorer® Green Fluorescent Protein
Purification Kit Catalog number 166-0005-EDU
CityLab’s SummerLab 4
IV. Rinse the Column with Wash Buffer
The wash buffer has a salt concentration that is lower than the binding buffer. It will
wash away the weak hydrophobic proteins. GFP is a strong hydrophobic protein.
Notebook Entry: Predict whether GFP will be washed off the beads by the wash buffer
or remain bound to the beads. Refer to the information in the diagram on page 1 for
help.
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Place the column in collection tube #2.
Gently add 250 µl of wash buffer to the column.
Drain the column into collection tube #2 until the buffer level is at the top of
the beads.
Notebook Entry: Was the GFP washed off the beads?
V. Release the sample from the beads
A solution without salt will remove the ability for hydrophobic proteins to stick to the
beads.
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Place the column in collection tube #3.
Gently add 750 µl of TE buffer to the column.
When the GFP begins to exit the column, collect the GFP in test tube #4.
VI. Clean the column
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Add 8 ml of TE buffer to the column.
Allow the TE buffer to drain to the top of the beads.
Add 2 ml of 20% ethanol to the column.
Replace the cap and top on the column.
Adapted from BioRad Biotechnology Explorer® Green Fluorescent Protein
Purification Kit Catalog number 166-0005-EDU