Fundamentally, you have two options: either measure the binding of a concentration series of both compounds in the absence of the other, or you measure competition between the compounds for the protein. The former method is generally simpler, the latter is useful mainly when you can detect the binding of one, but not the other compound (e.g., you have only one compound labelled, say, with radioactivity).

There are many methods available to detect binding, which one you use depends on the apparatus available, the stability of the complex and personal preference, however, you should try at least two methods to make sure the results agree. Methods include:

• Equilibrium dialysis: the ligand can pass a semipermeable membrane, the protein cannot. Then the concentration in the protein compartment will be the sum of bound and unbound ligand, in the protein-free compartment only the free ligand is present. Then bound ligand is calculated from the difference between the compartments. Beware of Donnan-effects when ligands are charged. Continuous dialysis is a variation of the method, [L] in the buffer is changed repeatedly and the appearance of L in the protein chamber is followed, useful mainly when [L] can be measured online, without taking samples.
• Ultrafiltration: similar to dialysis, but you use a centrifugal ultrafiltration device. The retentate will be bound+free, the filtrate only free ligand. doi:10.1006/abio.1998.2854
• Gel filtration: The column is equilibrated with ligand, then the protein sample is added. As the protein leaves the column, [L]t increases. doi:10.1016/0006-3002(62)90124-5
• Ultracentrifugation: The Chanutin et al. method spins a mixture of ligand and protein at such a high force that the protein concentrates at the bottom of the vial. Then measure both [protein] and [ligand] in several fractions of the gradient (http://www.jbc.org/content/143/3/753.full.pdf). In a plot of [L]t vs. [P], the intersection with the y-axis gives [L]f. In the Draper & v. Hippel's method the protein is spun through a sucrose gradient containing L. In effect, this method works like gel filtration (doi:10.1021/bi00572a003). Steinbach & Schachman's method measures both [L] and [P] continuously during analytical ultracentrifugation (doi:10.1021/bi00876a003).
• MS after ESI measures the appearance of [PL] due to the mass difference (doi:10.1006/abio.2000.4975, 10.1021/ac0346757, 10.1006/abio.2001.5062).
• If the complex is stable long enough, spin columns work very well. A tuberculin syringe is filled 2/3 with a matrix that absorbs the ligand, but not the protein (usually gel filtration, but IEC may also be used). The sample is spun through the column in a table-top centrifuge, the flow through contains only [L]b. I have used this method to measure binding (occlusion) of Na and Rb to Na/K-ATPase.
• Filtration through PVDF: The sample is filtered over a PVDF membrane, which retains protein and bound, but not free, ligand. If the PL-complex is stable long enough to survive washing of the membrane with ligand-free buffer, this method is very simple and quick. I have used it to measure ATP-binding to Hsc70, and to determine competition between ATP and its analogues for this enzyme.
• Native electrophoresis may also be used to separate PL from free L.
• Microscale thermophoresis, isothermal titration calorimetry, dual polarisation interferometry, quartz crystal microbalance and surface plasmon resonance require specialised equipment, but can give very good results with suitable samples.

Engelbert Buxbaum Wow, that was super comprehensive! Thank you for your information and help, it helped quite a lot! Thanks

Surface plasmon resonance