 Assalamu alaikum students. Today we are going to conduct a very important experiment. It is about protein identification. The title of the experiment is Vestrum Blotting. You know that identification of protein has importance not only in diagnostics but also in basic research. The basic and fundamental principle of Vestrum Blotting is very simple. You identify protein of your interest through its interaction with an antibody. So you design an antibody which is specific for binding with your protein. In this way if that antibody binds with your protein you will get the signal which will indicate that your protein is present. And if the antibody is not binding because there is no such protein in your sample then it will mean that your protein is not there. Also this experiment tells you about the down regulation, the over regulation that is over expression or under expression of your protein of interest. So if your protein is being produced in a very lower amount the signal produced because of interaction between the protein and the antibody will be very very dim. But if the protein is being overexpressed that is if it is being produced in a larger amount the signal because of the interaction between the antibody and the protein will be quite thick. And you see that thickness in terms of a band which we will show you later on. So students you can see here is our gel which contains our proteins. Now we will add some more filter papers above it. These filter papers are well soaked in the transfer buffer. So we will place them over it like this. And any presence of any bubble impossible here it has to be removed. No bubble should be there because if a bubble is there the current flow will be interfered. So we just press with this presser and take out if there is any bubble yes so fine. Now we have the beneath most the lower most layer of filter papers then there is a membrane then there is a gel and above the gel again there are filter papers. Now we will run the transfer from now. Now this is the lid which we have to put over this machine. Now we will connect the electrical terminals this electric terminal. You have to be careful so that the positive electrode is on positive one and the negative negative one. Well that's fine that's fine. Now we will adjust the voltage. I put the voltage on 20. I put the time on 30 minutes and let's now start it. That's fine. Now we can wait for 30 minutes and with this this important part of transfer is completed. Let's see how much protein or how well the protein is transferred. We will see it after 30 minutes. Okay guys now we will wait for 30 minutes till the transfer is complete and we will see how much our transfer has been successful after 30 minutes. So students you can see the transfer has been completed 30 minutes have gone. Now let's see our membrane. So now we will just let's turn it off. We don't need it anymore. So we remove the terminals and carefully open the lid. Fine you can see here is our gel. These are the filter papers we don't need it anymore. So let's see now how our transfer went. Let's remove this gel now. It's you know very flaccid so we have to be very careful. Yes now you can see here our membrane is very I mean it can show the transfer is very healthy very successful. You can see the proteins it looks that proteins are transferred very well because you can see the ladder bands here also. Now let's put this membrane into milk. Milk here acts as a blocking agent. It is used for removing or inhibiting the non-specific binding so that our target protein is distinctly identified. So we will put it over here in the milk and we will put this membrane on a shaker and we will let it to be shaked for around one hour. So now one hour is complete so let's stop it and now we will add primary antibody onto our membrane. So this tube contains our primary antibody which is dipped into the blocking buffer again which contains milk. We will put it onto the rotator and it will then be blocked for along with the primary antibody for overnight. I mean if you want faster sometimes you can also put it for one or two hours but it's better to put it overnight if you really want some very good results. Okay as you can see we have blocked our membrane and now we will put it into a solution which contains our primary antibody. Now this contains this is again the primary antibody is actually we have dipped it into the milk itself. I mean it's entered into a blocking buffer. Again the purpose of the blocking buffer is to inhibit the non-specific binding or at least minimize the non-specific binding. Now this is the primary antibody and now we will let it to be shaked overnight so it will keep on mixing for overnight. You can I mean if you in a hurry and you want urgent results you can put it for one or two hours also but ideally it is always good to put it overnight. Keep in mind but keep in mind that's very important. When you're going to put a membrane containing your proteins for overnight mixing with your primary antibody you have to keep in mind that the shaker is at 4 degrees C so that the whole process is taking place all this mixing process takes place overnight. The reason is that at room temperature there is every possibility that your protein gets denatured. Your antibody gets denatured the binding can be affected so it's always recommended if you have an overnight mixing then you should put your membrane at 4 degrees. Let the membrane to be into the solution and let it be mixed for overnight. Fine but for your convenience only we are just keeping it at room temperature just to show it for the sake of clarity and convenience and we are putting it over here. But student very important thing here I want to tell you that of course we are putting it overnight but this overnight processing should process should take place I mean at room not at room temperature but at 4 degrees C. So you put this shaker at 4 degrees C somewhere where 4 degrees C temperature is maintained and then let it be mixed there for overnight. We are showing it to you here only for your convenience but for your own purpose whenever you will be doing it you have to put the shaker at 4 degrees C if you are doing it overnight if you are doing it for one hour then it's fine at room temperature but for overnight you have to put it at 4 degrees C thank you. Okay guys now let's stop it because the primary antibody step is complete now let's now take this membrane out and put it into a wash buffer this is a wash buffer and let it to be shaked again for 10 minutes and guys you will repeat this process three times at least 10 minutes each 5 to 10 minutes it depends upon I mean what sort of primary antibody you are using so 5 to 10 minutes three times you will wash it after washing it we will add it put a secondary antibody to it we will now keep it on moving for around half an hour okay guys now the secondary antibody treatment is finished because this half an hour has just finished now now let us put it into a wash solution again and let's wash it in the same way as we wash in case of primary antibody wash it three times five to seven minutes each so guys we will now treat our membrane with chemiluminescent solution for exposing it for the purpose of detection of proteins this is chemiluminescent solution so you have to gently add it onto the membrane depending upon the size of the membrane you can add it accordingly now we can a little bit mix it like this so that it is homogeneously distributed and now we will put it into the chemiluminescent machine for the detection of the signals so guys you can see in this figure this thick band this thick band is representing our protein if you see the corresponding part of the ladder it shows that it's our protein because by the way our protein is fermenting to this the size of the band is corresponding to the protein so we are and there is no other non-specific band so we are very sure that this is fermenting to our protein of interest and in this way you can see how by the use of western blood we can identify our protein of interest not only this if let's say we have treated our sample with any drug we could see by the strength of the band whether or not this drug is over leading to the over expression or under expression of fermenting to