 We have been discussing about the use of surface plasmon resonance technology in different applications. As discussed in the previous lecture, SPR is increasingly being used in drug discovery and validation studies using small molecules. SPR biosensors offer exceptional sensitivity and high quality kinetics enabling the most challenging assays of analyzing the low molecular weight compounds with target molecules. It allows determination of broad range of affinity, different parameters like off-rate and on-rate constants. The ability to work with samples containing organic solvents like DMSO is a challenging task which is now supported by these SPR biosensors. In our previous lecture, we have screened the binding of 8 different small molecules to carbonic and hydrates and serum albumin and the results obtained were evaluated. The assay performance was studied and binding response at active and reference surfaces were examined. One of these drug molecules, Fluoresomite dissolved in DMSO from the previous screening assay will further be characterized for rapid kinetic analysis with carbonic and hydrates as immobilized target using a standard compound concentration series and DMSO solvent correction curve. We will now proceed with the immobilization and kinetic characterization of a small molecule with target protein. Let us have the experimental demonstration today. Now we will set up our immobilization for the kinetic template for this kinetic experiment. We will have the carbonic and hydrates immobilized by a mine coupling on a flow channel 4 so the target level of 1500 are used and we leave immobilization as 0 on flow cell 3. We go to the next tab, prime before run is highlighted here, analysis temperature at 25. Now we will have the following reagents prepared for the kinetic analysis of the interaction of carbonic and hydrates with various small molecules. Once the template is prepared, we go on to the next tab and we identify the run time of 36 minutes with a buffer consumption of at least 100 ml for this particular session. As per the volume listed on the table, we will prepare the buffers and reagents required for immobilization of carbonic and hydrates on the sensor chip. The ligand is prepared at a working concentration of 20 microgram per ml using an immobilization buffer of 10 millimolar sodium acetate pH 5. We will be using 1.05x PBS buffer as the running buffer which will be connected to the system followed by priming. EDC and NHS are prepared as suggested in the amine coupling kit for surface activation. 1 molar ethanol amine HCL pH 8.5 is prepared for blocking the free ester groups on the surface. Lastly, 50 millimolar NaOH was prepared which will be used to remove the electrostatically bound ligand. After preparation of these samples and reagents, we transfer the sample tubes to the appropriate rack and start the run as shown for beta 2 microglobulin immobilization in earlier lectures. We will hence look at the immobilization results obtained from this run. It is recommended to use lower immobilization levels for kinetics assay compared to screening assays. We targeted an immobilization level of 1500 RU for carbonic and hydrates. As shown in the figure, the immobilization procedure resulted in final immobilization level of 1283 RU. With this, we proceed further with setting up of kinetic assay wizard for low molecule weight compounds to study the interaction between carbonic and hydrates and furosimide. We now see how a small molecule kinetic template is set up. Similarly, here as well we have the startup, we have a sample flown at least once on the surface and a solvent correction with 8 different solvent correction points set here. Here also in the variable setting or in the cycle type, we go to the low molecular weight kinetic wizard. Check that the type is high performance, contact time is 60 seconds, dissociation time is 120 seconds, flow rate at 30 microlitre per minute, flow path both, extra wash with 50% DMSO, immobilization time of 30 seconds is provided here. Also there is a carryover control injection will be given at a flow rate of 40 microlitre per minute. Similarly, the sample solution is a variable here also the concentration and the molecular weight are variables here. So we go into the verification mode and see that the method has been verified and can be used to set up pair run. We go into the setup, here the flow path is 4 minus 3, we go on next we have 5 startup cycles, one set of solvent correction before the run and another select solvent correction after the run. In between we have the furosimide injected at various concentrations on the surface of the carbonic anhydrase and the kinetic data is accumulated here. We have highlighted prime before run and the following are the rack positions for various samples with furosimide at different concentrations, 50% DMSO, buffer for the startup, solvent correction, 8 different solvent correction sample vials provided for a successful kinetic experimentation of furosimide binding to carbonic anhydrase. For the volumes listed in the table we prepare the following samples and reagents, we prepare a running buffer of 5% DMSO in 1xPBS, we will be preparing a total of 5 concentrations of furosimide starting from 20 micromolar, 10 micromolar, 5 micromolar, 2.5 micromolar, 1.25 micromolar, 0.625 micromolar, 0.312 micromolar and 0.156 micromolar using a 2 pole sievel dilution in the running buffer with 2.5 micromolar concentration in duplicates. The running buffer will be used as a zero concentration negative control as well as in startup cycles. A wash solution is prepared with 50% DMSO in water. Lastly, 8 freshly prepared DMSO solutions ranging from 4.5% to 5.8% DMSO are prepared in 1.05xPBS for solvent correction. We will now work on the reagents required for the kinetic assay to study the interactions between human serum albimine and furosimide. In this study we will again be using 5% DMSO in 1.05xPBS as the running buffer which will also be used for the initial startup cycles. As prepared in the screening assay we will again prepare 8 different solvent correct solutions ranging from 4.5% DMSO in PBS to 5.8% DMSO in PBS by mixing these 2 solutions in different ratios. To re-iterate we have numbered the fuse as 1 to 8 and added 400, 600, 800, 1000 and 1400 microlitre of 4.5% DMSO in PBS 2 to 8 respectively. Following this, 5.8% DMSO is prepared and puts the lid on and keeps it in dark till we are ready with all the other samples. 8 different concentrations of the analyte furosimide in the running buffer including 20 micromolar, 10 micromolar, 5 micromolar, 0.625 micromolar and 2 micromolar is micromolar. We will prepare this concentration series in two fold solutions using running buffer starting from 20 micromolar which means we will mix 200 microlitre of 20 micromolar protein with 200 microlitre of running buffer to get the 10 micromolar concentrations. We similarly continue the two fold solution to obtain the rest of the concentration series. One of the concentrations should be run in duplicate so here in this experiment we will be running 2.5 micromolar concentration in duplicate, 3 0 concentrations which will be nothing but the running buffer. In the required volume in the specialized tube we will now proceed to insert these tubes the start-up 50% DMSO and the solvent correction solution into the appropriate rack and then into the system to perform the protein form molecule screening act. We have now placed all the tubes inside the rack including the VORS solution, the furosimide solution system to start with our kinetic assay. We will now start with our run as demonstrated in our earlier lectures. Before starting with the data analysis on kinetics of the interaction between carbonic anhydrase and furosimide we would like to show the bar graph obtained from the sensor gram which represents the relative response for report point binding against cycle number for samples. As reflected in the graph the relative response displayed linear increase with increasing analyte concentration with consistently low response for negative controls shown by cycle 7, 12 and 17 and almost the same response was obtained for duplicate sample that is 2.5 micromolar shown by cycle 13 and 18. Let us now have a more detailed data analysis on the kinetics assay. Now we will look at the kinetic data generated from the interaction of carbonic anhydrase with furosimide. The data looks like this way with a sample, solvent correction and a start-up. Now to evaluate this data we have to go into the evaluation, add solvent correction and once the solvent correction data looks okay say okay and the data is now subtracted from the sensor gram. From this again we go into the kinetic affinity mode surface bound data is shown here with the different colors for furosimide concentrations. We say next and we can clearly see the data having some spikes. So using the mouse button we can highlight that range, subtract or remove the selected areas. Similarly we can do that here as well. Once the subtraction is done we can go further to evaluate in the kinetic mode and fit the data. So here on the quality control tab you see all of them green and there is no significant bulk contribution found and data is uniquely determined and the data points are the kinetic on and off rates are within the range of the machine. The report shows on rate, off rate, affinity constant or binding constant, rmax of 12 Ru, various injected concentrations, flow rate, differences in the refractive index of the sample and the running buffer, phi squared below 1 and u value below 25. So this data shows the interaction of furosimide with carbonic anhydrase is 9.8 into 10 to the power of minus 7 molar. With this we go to the next tab and analyze the data. Here with the one to one fit of the data the residuals or the differences from the black line to the colored line should fall within the green areas or at least within the red area. Here they are within the green areas and it looks absolutely good to proceed with this data. Similarly, other parameters like the standard error is provided here. With this we will conclude the kinetic information generated for the interaction of furosimide with the ligand carbonic anhydrase recorded at 25 degrees centigrade and solvent corrected per the DMSO presence in the running and the sample buffers. I hope by now you also got a good understanding of SPR based assays and to analyze the data obtained from these binding experiments. In the next lecture we will discuss the basics of another label free platform which is biolayer interferometry and its applications in protein research. Thank you.