 To completely understand the function of a protein it is important to look beyond its expression, pattern and identify its potential interacting partners and determine the interaction dynamics because most of the systems are not only governed with a specific molecule but rather how their entire interaction network is built. To study the interaction between two binding partners in the SPR experiment one partner is attached to the surface and other is passed over the surface in a continuous flow. The interaction of ligand and analyte is measured by the SPR instrument as a change in refractive index over time and response observed is directly proportional to the change in mass concentration close to the surface. In the previous lecture we have performed a lab session and completed the immobilization of anti-beta 2 microglobulin antibody on the chip surface of CM5 chip will now move forward and perform the binding analysis of anti-beta 2 microglobulin with beta 2 microglobulin protein. As I mentioned we are talking to you about the standard protein and antibody pair however the same steps same procedures could also be followed for protein of your interest. However experimental design considerations may have to be modified as per the protein and antibody pair requirements. Let us continue our lab session and watch how to perform the binding analysis of anti-beta 2 microglobulin with its protein in the lab setting. We will go into the second session now and we will use our immobilized chip for a binding experiment. So here we will first prepare a template for the binding experiment. A binding experiment actually deals with screening or a single concentration screen of different compounds on the immobilized ligand and look for its ability to interact with the target that is immobilized on the surface. Before we go ahead with the binding experiment let us understand some important considerations. During sample injection the analyte is injected over the surface with a constant flow and concentration. Analyte in the sample binds to the immobilized ligand on the surface, the mass on the surface changes and the response is recorded. After sample injection buffer flows over the surface to allow monitoring of analyte dissociation from the ligand. Regeneration as already discussed by Dr. Srinivas is the process of removing bound analyte from the ligand on the sensor chip surface after analysis of a sample. Efficient regeneration which means removing bound analyte without affecting the ligand activity is crucial to a successful assay. If the regeneration is incomplete or the binding activity of the surface is reduced the performance of the assay is impaired. The choice of conditions for regeneration is dictated by the stability and nature of the ligand and analyte. In today's binding experimental setup we will be using HEPIS EP plus as the running buffer. We will be preparing three different concentrations of beta 2 microglobulin protein which are 8.5 nanomolar, 42.5 nanomolar and 85 nanomolar for evaluating its binding with the antibody having 8.5 nanomolar concentration in duplicate. These three concentrations will be referred to as low, medium and high. At times between the sample and the sensor surface should be sufficient to give confidently measurable response levels without compromising screening throughput. Contact time of 1 to 2 minutes are usually sufficient for a binding experiment. Here we will provide a contact time of 60 seconds at the flow rate of 10 microlitre per minute with the dissociation time of 60 seconds. An ideal regeneration condition is the one where analyte response of the same concentration is constant after repeated injections. Today we will be using 10 millimolar glycine pH 2.5 for regeneration of the surface. We will now proceed with our binding experiment protocol. File, before making the template we will open the file wizard again binding analysis new. Identify the flow path as 2 minus 1 as we have done our immobilization on 2 minus 1. Chip will be cm5 that is docked already. We will not have ligand capture, sample and regeneration. We will go to the next tab. Here we are not using any conditioning cycle. We will start with the startup cycle. Startup cycles are cycles of buffer used for equilibrating the system. So here basically buffer is used as analyte. So we can type as h, h, b, s, e, p plus buffer and from the pull down menu we select three cycles. Generally for binding experiments three cycles are selected going to the next tab for setting up for binding. We need to specify the contact time as 60 seconds default flow rate of 10 microlitre per minute dissociation time of any minute or any second by default we can consider 60 seconds. The regeneration solution we would prefer here would be 10 milli molar glycine ph 2.5 with the default contact time of 30 seconds flow rate of 30 microlitre per minute and with no stabilization time. We go to the next tab. Here we need to fill in the name of all single concentration compounds. So here we would select our analyte as beta 2m just that we have one analyte we will take it in three different concentrations. So we will name as low beta 2m medium and beta 2m high. So low indicates lower concentration medium medium concentration high concentration and we will go to the next tab we will select prime before run and normalize is not required here because the chip is already immobilized and we will go with the default temperatures and we will go to the next tab. Here we will not select a microtiter plate and this is our rack positions for a binding experiment here we have at the C1 position HBS EP plus buffer for three different startups we have three concentrations of analyte high medium and low and we have the regeneration solution here which is 10 millimolar glycine ph 2.5 and we will prepare our solutions and start the binding experiment. We will now work on the reagents required for the binding analyses of anti beta 2 microglobulin with beta 2 microglobulin protein. We will be using HBS EP plus as the running buffer which will also be used for the initial startup cycles. We will delude the stock solution of protein that is 100 micrograms per ml in the running buffer HBS EP plus to prepare 100 microliters of three different concentrations that is 85 nanomolar, 42.5 nanomolar and 8.5 nanomolar which are referred to as high and low concentrations in the experiment. We will also include one zero nanomolar concentration in the experiment which will be nothing but the running buffer. For the regeneration of the surface we have prepared glycine HBL ph 2.5 as the regeneration solution. We have transferred all the solutions in the specialized view starting from the startup beta 2m concentrations starting from 85 nanomolar, 42.5 nanomolar, 8.5 nanomolar and zero nanomolar. The regeneration solution is placed in this glass vial. We will now insert these tubes into the appropriate rack and then into the system to start with the binding analyses of anti beta 2 microglobulin with beta 2 microglobulin protein. We will eject the rack now to insert new vials eject rack and take the plate out or the sample rack and we will fill it with the binding vials so the vial positions are filled with different samples as you can see on the screen the startup is here so beta 2m medium beta 2m low beta 2m high are put at their respective positions and a vial for regeneration of 10 millimolar glycine ph 2.5 is here now we close the rack and will be inserted in the sample compartment by ejecting the rack compartment inserting the plate next tab so again we need to do all these checks check the time the estimated runtime of 38 minutes we have sufficient amount of buffer and we will now start the experiment we will save this template as binding save now we will save the result file again as binding and now the experiment has started chose running binding analysis with an estimated time of 38 minutes system is priming now and once the finish of the binding experiment we will take a look at the data before we analyze the binding data from the experiment let us look at a typical sensor gram for binding between a ligand and analyte tensogram as shown here is a plot of response against time showing the progress of interaction this curve is displayed on the system during the course of experiment we observe the baseline followed by the injection of analyte which leads to increase in the binding response during the association phase just after the stop of the sample injection we observe report point which records the response on a sensor gram at a specific time averaged over a short time window this is followed by dissociation phase regeneration and then back to baseline we will now proceed to analyze the data obtained from binding of anti-beta to microglobulin with beta to microglobulin protein after finish of our binding experiment by double click on the file the file is open now you can see here from our binding experiment which shows all sensor gram here so here the green ones are our startup so we have set up three or five different startups and the red ones are our actual data from beta to microglobulin what we will do is we will highlight only our sample data and now you see the data for beta to m we go on to sensor gram adjustment report point on the y adjustment baseline and say okay now our data is baseline to zero if we want we can as well go on to color sample and we will see the different samples in different colors with the legend on one side of the screen here we have low medium and high concentrations of beta to microglobulin injected over anti-beta to m we could subtract or delete the regeneration area now we can see our different concentrations of beta to m with one of them in duplicate so there is definitely binding of the beta to m to anti-beta to m antibody in a dope dependent manner the data can also be shown in form of a bar chart with all our red startup runs and green as our sample runs here we will highlight the sample runs only and see cycle seven eight and nine are our low medium and high concentrations data which are dose dependent binding going to another tab we can see the relative responses of each molecule from the binding at four for the duplicates and medium at 29 and high at 52 with this we will conclude our binding session and we prepare now for a kinetics experiment I hope you are able to follow the lab demonstration sessions these protein interactions are identified using wide array of applications however what is also required is an understanding of the extent to which these interactions actually occur therefore performing protein-protein interaction studies and calculating their kinetic values becomes very crucial we will continue the lab demonstrations and our discussion on SPR experiments for the kinetics analysis in the next lecture thank you