 Hello students, we were discussing about different experiments which are related to the SPR. Determination of interaction kinetics is one of the most striking applications for SPR biosensors. Understanding the kinetics is essential to evaluate the strength of a biomolecular interaction. The label-free real-time detection allows the kinetics analysis to be performed by monitoring interaction as a function of time over a range of analyte concentrations. If you think about previously the microarray experiments versus the SPR experiment, you may realize that for many of the binding experiment, you can get an idea from the microarray experiment whether a binding happens or does not happen. So you will get yes or no answer. Whereas in case of SPR, not only you get an answer that a binding happens, but also you can measure its kinetics. You can measure on-rate, off-rate and you can look at the Kd which is dissociation constant. The dataset obtained is then fit to a mathematical model and leads to determination of kinetic parameters like association and dissociation rate constants. Affinity constants exhibit the strength of binding between the molecules and it can be derived either from the rate constants or from a steady state binding analysis. A kinetic analysis experiment can be set up in two different ways. First, it can be multi-cycle kinetics where each analyte concentration is run as a separate cycle regenerating the surface after sample injection or second it can be single cycle kinetics which runs a series of analyte concentration in one cycle with no regeneration between sample injections. Today we are going to perform the kinetic analysis for our standard protein pair which is protein beta 2 microglobulin with anti-beta 2 microglobulin antibody using multi-cycle kinetics. So let us continue with the lab experimental session. We will start with low immobilization protocol set up for anti-beta 2 microglobulin as a lower immobilization level will be beneficial in this kinetic interaction analysis. We have to redo our immobilization in this case now we are selecting flow channel 3 and selecting flow channel 4. Flow channel 3 will be a blank immobilization and flow channel 4 we will immobilize anti-beta 2 microglobulin at 1200 Ru per a kinetic experiment. Here again we will highlight prime before run and keep the sample compartment temperature at 25 degree centigrade and we will go to the next tab. Now we will add vials with the rack positions and the volumes. As per the volumes listed in the table we will prepare the reagents for immobilization in a similar manner as demonstrated earlier in lecture 33. We prepared a working concentration of 30 micrograms per ml of beta 2 microglobulin antibody from a stock concentration of 1 mg per ml. Using an immobilization buffer of 10 ml molar sodium acetate pH 5 we prepared HIPIS EP plus as the running buffer which was connected to the system followed by priming. EDC and NHS were prepared to be used in 1 is to 1 ratio for surface activation. One molar ethanol amine HCl pH 8.5 was prepared for blocking the free ester group on the surface. Lastly 50 ml molar NaOH was prepared which will be used to remove the electrostatically bound ligands. Here the above mentioned solutions transport them to appropriate sample vials, place them in the suitable rack and start the immobilization run as we did in lecture 33. To save time we shall now proceed directly to analyze the data obtained from immobilization of beta 2 microglobulin antibody. So this is a Blanc sensorgram. After immobilization we will see the reference surface is Blanc immobilized. Here you see the activation of the chip by EDC and NHS and blocking of the surface with ethanol amine. So this will be the Blanc immobilization. Now we will go to another surface or another flow channel. Going to the next flow channel FC4 highlighted. This is a sensorgram for active surface immobilization involving protein. To start with there is a pre concentration estimation of the required amount of the ligand washed with the sodium hydroxide activation of the surface with EDC and NHS mixture, pulsing of the protein for the required number of RU's and then block of the extra sites with ethanol amine. The immobilization level is actually determined from this level subtracted to this level. Now this will conclude our immobilization procedure. We have successfully immobilized the required level of ligand for our next experiment. We will now set up a wizard for kinetic analysis. To start with we have selected from the wizard kinetic template selected the flow path 4-3 chip type CM5 sample and the flow is to regeneration. Once we are set up with this template we say next and start up cycles are selected at 5 with HPS EP plus buffer the sample contact time of 180 seconds is selected flow rate of 30 micro litre per minute is selected dissociation time of 600 seconds is selected coming to the regeneration. The regeneration solution is glycine HCl pH 2.5 with the contact time of 30 seconds flow rate of 30 micro litre per minute stabilization time of 0 seconds. And we now go on to the next tab and fill in the name of the sample as beta 2 microglobulin with a molecular weight of 11800 and concentration with 0, 2, 4, 8, 16, 32 and the repeat of 8 nanomolar again we say next and prime before run is highlighted. Now we have the rack positions this way we have regeneration buffer here start up here with the samples here is kinetic assay experimental setup we will be using HPS EP plus as the running buffer we will be preparing a total of five concentrations of beta microglobulin starting from 32 nanomolar 16 nanomolar 8 nanomolar 4 nanomolar and 2 nanomolar using 2 pole serial dilution in the running buffer and 8 nanomolar concentration in duplicate. We will be using HPS as 0 concentration negative control for the reaction as well as for the initial start up cycles. For regeneration solution we will be using 10 millimolar glycine pH 2.5 we shall now proceed to set up our experiment for kinetic analysis we will now work on the reagents required for the kinetic interaction analysis between anti beta 2 microglobulin and beta 2 microglobulin once again we will be using HPS EP plus and the running buffer which will also be used for the initial start up cycles we will be using our protein stock concentration of 100 microgram per ml to prepare five different concentrations of protein including 32 nanomolar 16 nanomolar 8 nanomolar 4 nanomolar and 2 nanomolar we will be preparing this concentration series in two pole dilutions using the running buffer starting from 32 nanomolar solution which means we will add 300 microlitre of 32 nanomolar concentration by 300 microlitre of buffer to prepare the similarly dilution series for rest of the concentration one of the concentration that is 8 nanomolar will be run in duplicate and we will also include a zero nanomolar concentration in the experiment we will be using glycine HPL pH 2.5 as a regeneration solution all these samples and reagents have been transferred in the required volumes in these specialized tubes and will now be inserted in the appropriate rack and then into the system for the kinetic interaction analysis so from the template here we have prepared the sample rack with the different vials to start with we have start up placed at E1 position and we have the different concentrations of beta to M with a concentration mark from positions at C1 B1 to B6 so 32 16 8 4 2 nanomolar and again zero once we have placed them we will also have glycine 10 millimolar pH 2.5 regeneration solution put at the A1 position now we close the rack we go on the screen eject rack say okay we say next on the tab so it takes 3 hours 34 minutes with 200 ml of minimum buffer and say start you can see here it says running kinetics affinity takes 3 hours and 34 minutes with a priming of 6 minutes once the immobilization is done and the experiment is being run this is how the data will end here and from the data we can go to tools viacore t200 evaluation software so this is how the data is generated you see all the startup runs here and we go on overlay to select sample data now we go on tools sensor ground adjustment report point baseline again to the tools color by concentration now you can see all the data is differently colored you see the rate of association dissociation and regeneration with the legend of the different concentrations of beta to M injected here as well like binding we will right click on the mouse and cut away this portion and now this is our actual data with the different concentrations of beta to M injected on the surface to evaluate this data we can go into the kinetic and affinity mode select surface bound the data is shown here we can go to the here you can see the different cycle numbers different concentrations of the beta to M at a flow rate injected was 30 microliter per minute contact time of 120 second and dissociation time of 300 second we have selected 2 minus 1 surface the ligand that is immobilized these anti beta 2 microglobulin and the sample is beta 2 microglobulin or analyte interaction performed at 25 degrees centigrade now we go on to this and see next if at all there are unwanted spikes can be removed in this region or in this tab by simply highlighting that area and right click and highlight that unwanted region and say remove selection similarly we could take away these spikes by highlighting that area is the right click of the mouse and say remove selection now that there are no spikes we can proceed to fit this data in the kinetic mode as you can see on the tab now you see here one-to-one binding and parameters here fit here so we could place mouse on the fit and press start so data is fit with the black lines super imposing on the colored lines and the following information is generated if you see the quality control tab report tab the residuals tab and parameters each of them has data from this experiment to start with we go on to the control quality control tab and see here all the three are in green with kinetic constants are within instrument specifications are uniquely determined and no bulk contributions found we have learned from our theoretical sessions bulk contribution will be more if there is a mismatch between buffer and sample now we go to the next tab report you can see lot of information that is generated here the on rate is given here similarly there is an off rate rate and there is a affinity or a binding constant provided here the r max is also given the various concentrations that was used to set up the experiment are provided here flow rate for this experiment is provided here the change in the refractive index due to differences in the sample and running buffer are provided here and a quality control parameter like high square and u value are also shown so k d refers to the affinity of the two molecules which is 2.6 into 10 to the power of minus 9 molar or 2.69 nano molar affinity r max or a maximum number of binding sites available at 24 chi square of 0.0901 should be below 1 at least u value below 25 is always considered to be good we go to the next tab residuals the residuals should be aligned between the red lines preferably between the green lines and here you can see that the data is completely within green lines and that indicates the superimposition of the black lines on the colored lines is very close now we go on to another tab parameter and find lot of information which are nothing but the standard error calculated for the data with this we conclude the interaction of anti beta 2 m to beta 2 microglobulin at 2.6 nano molar affinity I hope that these lab demonstrations of different steps involved to study and analyze the biomolecular interactions would have helped you in better understanding the SPR and the workflow involved in the next lecture we are going to have a scientist from Pratay Memorial Center ACTRAC who is going to talk to you about applications of SPR technology in biologically relevant problems thank you