When binding ligands to NHS beads, the amount of ester content per unit weight of the beads is approximately 200 nmol/mg. For the binding reaction, 3 concentrations are prepared (ligand solutions: 0.1, 0.3, 1 mM) in order to determine the optimal concentration. The amount of bound ligands depends on the ligand properties, and varies approximately from several nmol to 100 nmol. An amount of several nmol to several tens of nmol is best for achieving purification results. An excessive amount of ligands results in steric hindrance and has a negative effect on affinity purification.
20 mg of beads are sufficient for 2 studies of the affinity purification conditions. A total of approximately 50 mg of beads are necessary in order to secure a certain amount of the purified target proteins or other substance after the study of the conditions.
When designing a ligand in order to introduce a functional group for the parent compound of the protein you seek to identify, because the protein which will be purified varies depending on the location where the function group is introduced, it is recommended that receptor identification tests be performed by binding multiple ligands at different linker binding locations on the same parent compound. In the event that there is no functional group capable of binding to the ligand, it is necessary to change the chemical structure of the compound itself.
Secondary amines can be bound to NHS beads. However when both primary amine and secondary amine are present, the reaction will occur selectively with the primary amine.
When the amount of bound ligands is large, the beads become hydrophobic and dispersibility decreases. Therefore the beads are stored in a 50% methanol solution, not in water.
The proteins can be actually bound to the ligands in order to verify binding. At a binding concentration of 0 mM, there is no protein binding. If the protein band increases as the binding concentration increases, then good binding has been verified.
Ordinarily the affinity for the ligands of the proteins to be affinity purified is expressed as a dissociation constant (Kd) of 10-6 M or below. If the affinity is too low, there is the risk of a higher level of background noise.
When ligands with a relatively strong bonding strength are used, the results of studies by our company show a purification efficiency of approximately 50%. However the actual efficiency is highly dependent on the properties of the individual ligands and varies on a case-by-case basis.
The best beads are the NHS beads with activated COOH radicals on the COOH bead surface. The ε-NH2 radicals in the protein lysine residue bond with the COOH radicals on the bead surface. It is also possible to bind proteins to the epoxy beads.
It may be possible to bind the proteins by introducing a His-tag or by using cysteine residue sulfhydryl radicals (SH radicals).
If binding is performed when 50 mg of protein is supplied to 1 mg of NHS beads, approximately 10 – 50mg will be bound, although the result varies depending on the protein. It is possible to increase the amount that is bound by increasing the amount supplied.
The best beads are the NHS beads with activated COOH radicals on the COOH bead surface. The ε-NH2 radicals in the protein lysine residue bond with the COOH radicals on the bead surface. It is also possible to bind peptides to the epoxy beads.
There is no problem. However perform centrifuge separation before using to remove any impurities (such as degenerated proteins).
Serum proteins or extracts from cultivated cells or tissues can be used. When using cultivated cells as the protein source, 109 nuclear proteins or 107 - 109 cellular proteins are required.
While it is not impossible, it is not easy to use affinity purification by this method for GPCRs and ion channels. There are cases when affinity purification can be used by solubilizing the membrane protein with a surfactant.
It is necessary to consider the optimal conditions by varying the concentration of ligand binding to the FG beads®, the salt concentration in the buffer, and the surfactant concentration. This can sometimes be improved by carefully performing dispersion when screening. Insufficient masking is also a possibility, so it is necessary to suitably mask the functional groups which are not bound by the ligands.
Change the buffer composition and detect a more highly specific band. Alternatively, it is necessary to perform competitive inhibition tests and drug elution, and verify whether or not the band is specific to that ligand.
There are no problems with using TBS in place of HEPES, and NaCl in place of KCl.
This is because bonds are divided between somewhat weaker bonds (which can be broken by salt) and strong bonds (which can be broken by a sample buffer plus heating). However there is no problem with performing boil elution alone.