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Suitable conditions for binding/elution
The problem of finding a suitable ligand in affinity chromatography is not restricted to just specificity, but concerns also the binding strength and the kinetics of the ligand-target molecule reaction.
Ideally the binding should be strong enough to avoid leakage during the sample application and wash phase, while the target should be completely released during the elution phase.
In other words, one has to find a ligand specific enough, but with a binding strength that allows safe elution.
Ideal affinity chromatography conditions also require the kinetics of the binding and desorption reactions to be fast enough to ensure complete reaction under normal flow rates.
Expected results under ideal conditions
Fig 4.1. No leakage during sample application and wash. Target molecule elutes as a narrow peak. |
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Binding
Reversible reactions such as the reaction
between the target molecule and the affinity ligand are characterized by their equilibrium dissociation constants (KD).
The smaller the value of KD the stronger the binding.
KD values for good binding are typically in the range of 10-4 - 10-6 M.
Fig 4.2. K D values for good binding are typically in the range of 10 -4 - 10 -6 M.
The KD value can be influenced by changing conditions like pH, ionic strength, temperature, polar properties etc.
KD values > 10-4 provide too weak a binding and the target molecule may "leak" as a diluted broad zone during sample application and wash.
Figure 4.3 Leakage of target due to too high a KD: With too low a KD, the target molecule elutes spontaneously and diluted during sample application.
If a ligand binds too strongly, it will be difficult to elute the target molecule without introducing harsh conditions. Under such conditions there is always a risk of abolishing the biological activity of the target molecule or finding it “irreversibly” adsorbed to the affinity medium. |
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Elution
Ideal elution requires the target molecule to desorb completely from the affinity ligand in elution buffer.
Two possibilities exists:
1.
Apply conditions (pH, ionic strength, temperature, polar properties etc.) that changes KD from low to high.
KD values suitable for elution are typically in the range of 10-1 - 10-2.
Fig 4.4. Elution (desorption): KD values suitable for elution are typically in the range of 10 -1 - 10 -2.
For KD values <10-2 retardation of the target molecule may occur during elution with severe peak broadening as a probable result.
Fig 4.5. Retarded elution due to too low a KD value: Too strong a binding may lead to retarded elution. 2a.
Add a free ligand (or analogue) to displace the target molecule by competitive binding to the target. Example: Elution of NADP dependent enzymes from Blue Sepharose by adding NADPH
Fig 4.6. Elution by displacement, free ligand: Free ligand is added to displace the target from the matrix-bound ligand.
2b.
Add a competitor to displace the target molecule by competitive binding to the matrix-bound ligand .
Example: Elution of HIS tagged proteins from
HiTrap Chelating by adding imidazole.
Fig 4.7. Elution by displacement, free target analogue: An analogue is added to displace the target from the matrix-bound ligand. |
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Binding/desorption kinetics
Ideally affinity chromatography provides distinct and rapid binding and desorption of the target molecule and the expected results should look like those shown in fig 5.1.
Fig 5.1. Expected behavior, quick on/quick off: With sufficiently fast binding and elution kinetics the target molecule elutes as a sharp peak.
If slow binding and/or desorption is experienced the best way out is to turn to another affinity system providing "fast on/fast off" kinetics, if available.
Sometimes, however, slow kinetics cannot be avoided and special ways to bind and elute the target molecule have to be adopted or recovery may suffer.
Slow Binding
Leakage of target molecule during sample application and wash (Fig 5.2) indicates that the sample residence time is too short for complete binding. If such leakage escapes attention the results might be wrongly interpreted as providing low recoveries.
Fig 5.2. Slow binding requires enough residence time for complete binding.
A way to extend the residence time is to inject the sample in small portions and stop the flow after each injection (Fig 5.3). The duration of the flow stop has to be worked out by trial and error.
Fig 5.3. "Stopped flow" binding: Increased residence time can be created by injecting the sample in small portions and stopping the flow between injections.
Slow desorption
The problem here is not the degree of purification obtained, but rather that the eluted target molecule may elute as a quite diluted long zone (Fig 5.4).
Fig 5.4. Slow desorption may result in broad and dilute peaks.
Even in this case an elution technique based on "stopped flow" may help.
One column volume of eluent is pumped into the column and the flow is then stopped. This will allow desorbed target molecule to accumulate in a restricted volume of eluent before leaving the column and thus elute in more concentrated form. The stopped flow elution is then repeated until all target has been eluted (Fig 5.5.).
Fig 5.5. "Stopped flow" elution: "Slow off" kinetics requires extra time for full desorption. |
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