Affinity chromatography is a versatile separation protocol that uses the biological interactions for characterization and detailed analysis of sample components. It is based on highly specific interactions between two molecules, such as the interactions between enzyme and its substrate, receptor, and ligand, or antibody and antigen. These reversible interactions are used for the purification by placing one of the interacting molecules, considered as affinity ligand, onto a matrix to make a stationary phase while the target molecule is run through the mobile phase. Highly selective nature of the method enables a fast, single-step purification of the sample components from several hundred to thousand-fold. Other applications of the technique include the study of drug or hormone interactions with the binding proteins, the ability to concentrate substances present at low concentration and to separate the proteins based on their biological function. The development of the affinity liquid chromatography has enabled the biomedical researchers to explore and investigate protein-protein interactions, post-translational modifications, and protein degradation.
The stationary phase of the affinity chromatography consists of a support medium (e.g., cellulose beads) on which the substrate is attached covalently to expose the reactive groups essential for enzyme binding. As the crude protein mixture is passed through the chromatography column, proteins with a binding site for the immobilized substrate bind to the stationary phase, while all the other components of the sample are eluted in the void volume of the column.
The affinity chromatography matrix is made of porous support materials such as agarose, polyacrylamide, polymethacrylate, cellulose, and silica. The matrix should be selected on the basis of physical and chemical properties. Spacer arms could also be incorporated between the matrix and the ligand for efficient binding and better binding environment. Ligands for the affinity chromatography are selected depending on the molecules to be separated. The ligands can be covalently immobilized or adsorbed on a surface via bio-interactions, entrapped within a pore, or conjugated with a metal ion. The isolated components can be taken for analysis from the derived elute.
- Centrifuge the affinity column for 5 seconds to set the resin at the bottom of the column.
- Open the cap and break the bottom plug of the column.
- Place the affinity column in a 2ml tube and centrifuge it for 5 seconds to drain the buffer out.
- Place the column in a 2ml collection tube and add 0.2ml binding buffer. Centrifuge the column for 5 seconds, discard the buffer collected in the collection tube. Perform this step twice to equilibrate the column thoroughly.
- Place the column in a 2ml collection tube.
- Carefully add 100μl in the column. Incubate it for minutes and then centrifuge for 5 seconds. Collect the flow-through in the collection tube and label this tube as Fraction 1.
- Wash the column thrice with Affinity Binding Buffer. Place the column in a new tube. Add 0.2ml Affinity Binding Buffer in the column. Centrifuge for 5 seconds and label it as Fraction 2. Perform this step twice in two separate tubes and label the tubes as Fraction 3 and Fraction 4, respectively.
- Elute the sample using a salt gradient: a mixture of the Affinity Elution Buffer and elution buffer with a high concentration of salt.
- Place the column in a clean collection tube. Add 0.2ml of the lowest salt concentration buffer. Centrifuge it for 5 seconds and collect the fraction.
- Place the column in a fresh tube and apply the next elution buffer starting with fraction 6 to 10 and repeat step 7. Collect all the 6 fractions in separate 2ml tubes.
Purification of DNA-binding proteins (Kerrigan. & Kadonaga., 2001)
- Add 440 μg of each oligonucleotide mixed in to make a total volume of 130 μl in a 1.5-ml microcentrifuge tube.
- Add 20 μl of 10× . Incubate it for 2 minutes at 88°C, 10 minutes at 65°C, 10 minutes at 37°C, and 5 minutes at room temperature.
- Half the mixture in separate microcentrifuge tubes. To each 75-μl aliquot, add 15 μl of 20 mM adenosine triphosphate (ATP) (pH 7.0), ∼5 μCi [γ-32P] ATP, and 10 μl of 10 U/μl T4 polynucleotide kinase (100 U total). Incubate it for 2 hours at 37°C.
- Add 50 μl of 10 M and 100 μl water in each tube to inactivate the kinase and heat for 15 minutes at 65°C. Allow the mixture to cool to room temperature.
Purification of phosphorylated oligonucleotides
- Add 750 μl of and mix by inverting the tube. Microcentrifuge it at high speed for 15 minutes at room temperature to get the pellet containing DNA. Discard the supernatant.
- Resuspend the pellet in 225 μl .
- Add 250 μl of phenol/chloroform/isoamyl alcohol (25:24:1 ) to each tube. Vortex for 1 minute. Microcentrifuge at high speed for 5 minutes to separate phases. Transfer the upper layer (aqueous phase) to a new tube.
- Add 250 μl of 24:1 chloroform/ to the aqueous phase. Vortex for 1 minute. Microcentrifuge at high speed for 5 minutes to separate phases. Transfer the aqueous phase to a new tube.
- Add 25 μl of (3 M) to aqueous phase and vortex.
- Add 750 μl of 100% ethanol and mix by inversion. Microcentrifuge at high speed for 15 minutes to pellet DNA. Discard the supernatant.
- Wash the pellet with 75% ethanol (800 μl) and vortex. Microcentrifuge at high speed for 5 minutes. Discard the supernatant.
- Dry the pellet using the vacuum evaporator.
- Add 65 μl water and 10 μl of 10× linker/ to the derived pellets.
- Dissolve the DNA by vortexing.
- Add 20 μl of 20 mM ATP (pH 7.0) and 5 μl of 6000 U/ml .
- Incubate for 2 hours at room temperature or 15°C overnight.
- Monitor the ligation reaction on agarose gel electrophoresis, using 0.5 μl of ligation reaction per gel lane. Visualize the DNA with the help of ethidium bromide staining and UV photography.
Purification of oligonucleotide multimers
- Add 100 μl buffered phenol to the 100-μl of ligation reactions and vortex it for 1 minute. Microcentrifuge at high speed for 5 minutes at room temperature. Transfer the aqueous phase to a new tube.
- Add 100 μl of chloroform/isoamyl alcohol (24:1) to the aqueous phase. Vortex for 60 seconds and microcentrifuge at high speed for 5 minutes at room temperature. Transfer the aqueous phase to a new tube.
- Add 33 μl of 10 M ammonium acetate to the aqueous phase and vortex it.
- Add 133 μl and mix by inverting the tube. Incubate for 20 minutes at -20°C. Then, microcentrifuge at high speed for 15 minutes to obtain DNA in the pellet. Discard the supernatant.
- Add 225 μl TE buffer and vortex to dissolve the pellet. Add 25 μl of 3 M sodium acetate and vortex again