- No attempt is made to describe the routine operation and maintenance of the BIAcore, as this is comprehensively described in the BIAcore instrument manual. - Fortunately, an adequate working knowledge of the technique does not require a detailed theoretical understanding. - Importantly, a response (background response) will also be generated if there is a difference in the refractive indices of the running and sample buffers. - A second, related problem is that, following dissociation of the analyte, it can rebind to the unoccupied ligand before diffusing out of the matrix and being washed from the flow cell. - bind to the native protein are an excellent means of assessing the structural integrity of the recombinant protein. - a useful rule of thumb is that an interaction should reach 99% of the equilibrium level within 4.6/k off seconds. - This upper limit is dependent on the size of the analyte. - It is then simple to evaluate the effect of the mutation on the binding properties (affinity, kinetics, and even thermodynamics) of the immobilised protein. - The angle of the minimum reflectance ( θ spr ) is calculated by fitting a curve to all this data, thereby enabling θ spr to be measured at a far greater resolution than the resolution at which the data are actually collected.. - The θ spr is measured along a section of the sensor surface rather than at a single point. - While slight shallowness of the dip is acceptable, and is common after coupling large amounts of protein, more severe abnormalities should not be ignored. - The most challenging step when setting up SPR experiments is immobilising of one of the proteins (the ligand) to the sensor surface without disrupting its activity. - Firstly, because they usually have multiple copies of the functional group that mediates. - Evaluate the activity of the immobilised protein (Section 4.5).. - They can therefore be used for any of the sensor chips that have such carboxymethyl groups (See Table 2). - proteins net positive charge will decrease and because electrostatic interactions will be screened by the high ionic strength of the running buffer. - Incomplete dissociation suggests that the interaction was not purely electrostatic, perhaps because of the binding, at low pH, of a denatured form of the protein.. - The structural integrity of the protein should be evaluated (Section 4.2.6) before regeneration is attempted. - The goal here is to elute any non- covalently bound analyte without disrupting the activity of the ligand. - If this fails to elute >90% select a stronger regeneration buffer of the same type and return to step 4.. - The best way to achieve different levels of coupling is to change the duration of the activation step, by varying the volume of NHS/EDC injected. - The level of coupled ligand varies in proportion with the duration of the. - It is important to evaluate the functional integrity of the immobilised protein. - It is preferable to check the binding of several mAbs, including ones that bind to the same binding sites or, failing this, the same domain of the natural ligand. - Intact mAbs are less useful because of the uncertaintly as to their binding stoichiometry.. - The extent to which the analyte needs to be characterised depends on the nature of the experiment.. - Quantitative measurements (Section 7) require that the analyte is very well characterised and of the highest quality. - In order to determine affinity and association rate constants it is essential that the concentration of the injected material is known with great precision. - Firstly, it is necessary to determine the. - Although this can be calculated from the primary sequence it is best. - determined directly by amino acid analysis of a sample of the protein with a known OD 280 . - Secondly, it is important to assess what proportion of the purified protein is 'active', i.e. - It is important to emphasise that. - Fortunately the presence of multivalent aggregates is readily excluded by analysis of the binding kinetics (Section 7.2). - Quantitative measurements are far more demanding than qualitative measurements because of the quality and amount of materials required and the difficulties associated with designing the. - The ‘association constant’ (K A ) or affinity constant is simply the ratio at equilibrium of the. - Many prefer to express affinity as the ‘dissociation constant’ or K D , which is simply the inverse of the K A , and therefore has the units M.. - However, because of the difficulties associated with obtaining definitive kinetic data on the BIAcore, equilibrium binding analysis is more reliable. - Where captured ligands dissociate spontaneously or require regeneration, it may be difficult to maintain the level of the ligand constant.. - It is important to ensure that the analyte injections reach equilibrium. - This should be checked by showing that a reference analyte binds to the same level at the beginning and end of the experiment. - The same affinity should be obtained irrespective of the order of injections.. - The K D and Max values are best obtained by non-linear curve fitting of the equation to the data using a suitable computer software such as Origin (MicroCal) or Sigmaplot.. - A Scatchard plot of the same data (see Chapter 3), obtained by plotting Bound/C A against Bound, is useful for visualising the extent to which the data conform to the Langmuir model. - The affinity should be also be confirmed with two independently-produced batches of protein and with different recombinant forms of the same proteins.. - cooperativity between binding sites or self-association of the analyte, either in solution or on the sensor surface.. - The shape of the Scatchard plot will also depend on the surface density of ligand. - Determine accurately the concentration of the analyte and the proportion that is active.. - Obtain a rough estimate of the K D by injecting a series of five-fold dilutions.. - Repeat the injection of the control sample.. - Use different recombinant forms of the same proteins.. - For the simple 1:1 model binding will reach 99% of the equilibrium level within 4.6/k off seconds. - Similarly, it will take 4.6/k off seconds for 99% of the analyte to dissociate. - The period during which analyte is being injected is termed the 'association phase' whereas the period following the end of the injection is termed the 'dissociation phase'. - Because of the high surface density of ligand on the sensor surface, the rate at which analyte binds ligand can exceed the rate at which it is delivered to the surface (referred to as mass transport). - Convection transport can be. - However re-binding will still occur because diffusion out of the unstirred layer is little affected by convection transport. - Finally, when the ligand is saturated the initial part of the dissociation phase will not be affected by re-binding, since no free ligand is available for re-binding. - However such selective analysis of a part of the dissociation phases should be avoided. - In summary, mass transport limitations, which lead to an underestimation of the intrinsic kinetics, are aggravated by low flow rates, high levels of immobilised ligand, and high intrinsic association rate constants. - Determine accurately the concentration of the analyte.. - Prepare a two-fold dilution series of the analyte ranging from concentrations of 8*K D to approximately 0.25*K D . - The duration of the injection is not critical since binding does not need to reach equilibrium. - Subtract the response in the control flow cell from the responses in each of the ligand flow cells.. - Attempt a global fit of the simple 1:1 binding model to the entire series of curves. - fit as much of the association and dissociation phase as possible.. - If this is not possible the measured rate constants should be considered to be lower limits of the true rate constants (16,18).. - If possible, confirm the results using different recombinant forms of the same protein.. - A second important point is that more of the analyte is needed for kinetic analysis. - For any particular sensorgram as much of the data as possible should be included in the fit. - dissociation phases, omitting only the 'noisy' few seconds at the beginning and end of the analyte injection. - A rigorous test of the binding model is to fit it simultaneously to multiple binding curves obtained with different analyte concentrations. - An important internal test of the validity of the kinetic constants is to determine whether the calculated K D (K Dcalc = k off /k on ) is equal to the K D. - The binding stoichiometry can be determined if the molecular mass of ligand and analyte are known and the activity of the ligand is known. - A fit of the simple 1:1 binding model to this data yields the maximum level of analyte binding as well as a K D . - The key problem is establishing the activity of the immobilised ligand. - If it is assumed that ∆H and. - ∆S° are temperature-independent, the linear form of the van’t Hoff equation can be used.. - The ∆C p is a measure of the dependence of ∆H (and ∆S) on temperature. - This negative heat capacity is believed to be the result of the disruption at high temperatures of the ordered 'shell' of water that forms over the non-polar surfaces of a macromolecule. - ∆C p is a useful measure of the extent of non-polar surface that is buried upon binding (25). - The extent of this increase is a measure of the amount of thermal energy required for binding or dissociation, and is referred to as the activation energy of association (E a ass ) or dissociation (E a diss. - One of the most useful features of SPR is that it provides, through binding analysis, a quick way of checking the structural integrity of recombinant molecules. - If the surface of the glass is coated with a thin film of a noble metal (e.g. - some of the light is 'lost' into the metallic film. - It is a consequence of the oscillation of mobile electrons (or 'plasma') at the surface of the metal film. - When the wave vector of the incident light matches the wavelength of the surface plasmons, the electrons. - The 'coupling' of the incident light to the surface plasmons results in a loss of energy and therefore a reduction in the intensity of the reflected light. - It is because the amplitude of the wave vector in the plane of the metallic film depends on the angle at which it strikes the interface that an θ spr is observed. - Because of this, the resonant frequency of the surface plasma wave (and thus θ spr ) depends on the refractive index of this medium. - This dislodges air-bubbles from the bottom of the container, helps ensure that the meniscus is horizontal.. - developments and includes a continuously updated list of SPR publications and an electronic version of the BIAjournal.. - Can be used with most proteins. - Biotinylation can be. - Rebinding • The fit gwill be worse at higher levels of immobilization.. - More than one form of the analyte binds to a single ligand.. - Dissociation will slow as the duration of the injection is increased. - Dissociation will not be affected by the duration of the injection. - However, in the reverse orientation dissociation will slow as the duration of the injection is increased.. - The dissociation phase will slow as the duration of the injection is increased