Affinity chromatography

welcome back friends welcome to another video from somos biology and in this video tutorial we'll be talking about affinity chromatography though I didn't read that we already talked about the size exclusion chromatography in the earlier videos you can watch that video so here we'll be talking about affinity chromatography affinity chromatography is very important type of chromatography which we use to separate proteins and also we can use this affinity chromatography techniques more in the immuno chromatographic applications you know where we need to separate different antibodies against specific infections to check samples serum so that the individual is affected or not so it has a very important applications for our life and detection of diseases so what is affinity chromatography you know in any kind of traumatic rafi we know of we run the chromatography in a column the column is consisting of two different phases one is the stationary phase which is solid or liquid may be solid or liquid in this case the phase is solid and in this case the solid phase is made with agarose okay normally it is made with any type of polymer like agarose or silica gel or whatever it is ultimately it should create a polymer structure and it should have pores in it now in this case the stationary phase is made with agarose and the other phase that is the mobile phase the mobile phase in this case is also a solute the solvent in this case it's also aqueous solution that we can use in this case okay so it's liquid in nature so we have this two thing stationary phase is agarose Network which is solid mobile phase is liquid that can be say that sample serum that is the sample in this case we can use the patient blood serum so now let us talk about it so in the column we have the stationary phase fixed and then we add the specimen or the sample and those liquid sample will slowly move through the column okay and among that solution you know the idea of chromatography is to separate molecules from a mixture of the molecules so here in this case we are we want to separate certain antibodies against a specific infection so we do not want any other proteins to be separated so how would you do that we can use a specific affinity interactions between molecules to separate them from each other how that is the fund of affinity chromatography affinity means there are certain molecules who have affinities with each other I give you some example some example is antigen for a specific disease it could be the breakdown material of virus or bacterial body parts or DNA that can act as antigen or protein of bacteria or virus that can act as antigen and that can interact with antibody specific antibody against that antigen this is an affinity towards each other they have a specificity so if they find themselves they will bind similarly we also have histidine histidine has affinity towards nickel cobalt these are the ions where the histidine is very very fond of our very affinity towards so if histidine is present and there are nickel and cobalt ion is present history is going to bind with the nickel and cobalt another thing I'll give you GST glutathione is transferase which has the affinity towards glutathione okay so these are the idea so this is how the affinity works these are the three different examples of three different molecular affinities with each other GST affinity towards good glutathione histidine affinity towards nickel or cobalt antigens have affinity towards a specific antibody so we can exploit this affinity of molecules with each other to separate them how now let's draw the column if I draw the column here there could be stationary phase the stationary phase is made with the agar as I told you but the agarose will be attached with a bait it's just like catching a fish with a bait you can catch a fish so here in this case the bait that we use is either of these molecules for example in case of the imac the chromatographic process is called as iMac okay in this iMac chromatography where we use ion exchange system for attaching a specific molecules for example here with this iMac type of chromatography agarose is attached with nickel or cobalt so here in the stationary phase what we have we have we have this let us say let us let us draw it in a way that we will understand it's actually fully covered the the stationary phase is completely covered through the through this chamber but I am drawing it in small part so that you will understand say this is the agarose and the agarose is interacting with the nickel bulk or cobalt these are the nickel for example okay these are attached nickels and now we will apply the mixture of proteins there will apply the mixture so once you apply the mixture of proteins through this chamber they will start to come down using the gravitational force to the bottom now once this process is going on if some of them contains this histidine as a amino acid sequence as a tag those protein molecules are going to bind histidine in rest of the protein histidine rest of the protein and rest of the proteins which will not carry histidine will simply come out they will not bind so remember only those molecules with histidine tag will bind to the nickel rest of them will not bind so they will be washed through using the buffer so let us say this process is done we use the buffer to wash the unbound protein molecules so rest of the unbound molecules are simply washed away so now what we have we only have the column which is attached to nickel and now histidine tagged molecules are attached to them so now they are attached or bound like this after this process is done the final stage of affinity mcgrevey like any other column chromatography is the illusion right we need to take out our target molecules from the column so here the illusion will be done using either changing the pH or changing the salt concentration okay now if you are using for histidine and nickle interactions we use emitters or in this case Amida's ol can help this histidine tag protein molecules to come off from the nickel because the emitters or group containing molecules are going to tend to bind to the nickel and that will kick this histidine tag proteins out from the previously bound state okay so you see me does all type of molecules here for illusion of histidine tag proteins from this nickel chromatography here I max full-form is immobilized metal ion affinity chromatography and this is the example of the imac that we saw where we use nickel or cobalt as immobilized metal ion and then we use the affinity towards nickel which is histidine to separate specific protein from a mixture of proteins that is an affinity chromatography so immobilized metal ion affinity chromatography or imac this is one of the examples now in other cases also we have antigen antibody separations we can use it for this GST glutathione separation now this type of technique is very much useful in case of recombinant DNA technology and recombinant protein purification because once the recombinant proteins are made we tend to attach specific tag molecules tag regions like GST tag or like hexa histidine tag so we have six histidine residue consecutively that will be considered as hexa histidine as tagged and we can also put some gene sequences for the GST production so what we know is that after the molecules will be prepared after the recombinant DNA technology those proteins of interest will either carry GST or carry histidine with them so from mixture of other cellular proteins that come from that same cell we can easily we take these molecules out this proteins out from the mixture of other proteins using this affinity chromatography techniques that is why it is very very important it is vigorously used in all the expression vector studies and expression analysis of certain proteins and genes that we know another very important example is the detection of antibody now this is also known as immuno affinity chromatography now the immuno affinity chromatography means the chromatography affinity chromatography is used in immunological perspective now how does this thing happen in this case of community chromatography we deal with let us say a particular disease when it occurs in our body antigens are present now in response to that antigen our body produces antibody now specific antibody will interact with that antigen with antigen antibody interaction with great affinity towards each other so now we know if we find the specific antibody that can pair with the antigen it is going to help us to track down the disease very well so that antibody we need to find out and we can find out an antibody by selecting for the antigen now we know for a specific disease antigen X for example is responsible so what we do we take antigen X we attach antigen X with the agarose molecule here attacked with the stationary phase molecule so it will be instead of in earlier case we saw it is attached with the nickel but instead of nickel let us say we attach it with the with an antigen molecules let us say these are the antigen molecules that we build now we allow we apply the serum the patient's serum because you know if antibody is produced it should be produced against a specific antigen it is found in the serum the blood serum so you have the blood serum now if the antibody is present then the antibody is going to bind with this antigen from the serum right and after the wash off stage and then the final dilution stage in the Aleut end if we find the antibodies are present that means that person is having that particular disease caused by the antigen so we can track down which disease the person is having using this affinity chromatography it is known as immuno affinity chromatography this is very important technique that we can use another very important applications of affinity chromatography is to separate a specific you know most trans translational modification which is known as the glycosylation glycosylation means once the proteins are produced the proteins are modified in the golgi apparatus the different types of groups are attached methyl group glycosylation means addition of carbohydrate molecules to the protein either with the in terminal linkage most of the time the in terminal part of the amino acid is attached to a specific type of carbohydrate molecules so that is known as the glycosylation process now this glycosylation of all those proteins after the pro translation is known as post translational modification and after this post translational modifications once we produce all those type of proteins sometimes it is important to only figure out the glycosylated proteins they are also known as glycoproteins okay so sometimes we need to figure out we need to take out the glycoproteins from the mixture of the total proteins of cell so remember so we have glycoproteins and we have only proteins okay so how you figure out which are the glycoproteins and which are not let's say you track the cell open and you take all the protein contents it will include the glycoproteins it will include the other proteins out there the structural proteins and stuff everything will be there now from the mixture you only want glycoproteins to be fished out so how do you do that we can use a specific type of affinity chromatography to fish out glycoproteins it is known as lifting affinity chromatography because these glycoproteins that means the those carbohydrate regions or carbohydrate moieties have an affinity towards a molecule known as lectin lectin can interact with those carbohydrate more it is of a protein so whatever protein contains those carbohydrate moieties can interact with electing can bind with letting okay and so in those case what we have we have the column and in that column we have those let us say this is the column and in that column I am again drawing in a small part of it and it is paired with the lectin and we know the leg thing has the affinity towards the those carbohydrate moieties of the protein so whatever move protein with the carbohydrate moiety let us say these are the proteins with carbohydrate moiety it will bind to the lectin but other proteins which do not have any carbohydrate moiety will not bind so they will come out easily so again from mixture of two different modes or types of proteins we can figure out which are glycoproteins and which are not and then we elude the glycoproteins again illusion can be done based on changing in pH usually we drop the pH down we can use glycine glycine buffer of pH 2.8 which is pretty much acidic you know two point H pH so we can use this glycine buffer with at 2.8 pH that can help in separation of those bound glycoproteins from the lectins and that we can separate molecules from each other so that is another example of affinity chromatography electing affinity chromatography so these are the different examples of chromatography affinity chromatography technique and this is really really good in very applicable use it ok so that's all about affinity chromatography guys if you liked the video please hit the like button share this video so that it can help your friends also and also definitely subscribe to my channel because this is the way to grow it so hit the subscribe button and join our community thank you

Channel: Shomu's Biology

Views: 153,625

Keywords: suman bhattacharjee, shomus biology, biology online, biology courses, life science, biology major, purification, protein, affinity chromatography, chromatography, affinity, affinity chromatography reagents, chromatographie, protein purification, affinity chromatography his tag, affinity chromatography for protein purification, affinity chromatography procedure, affinity chromatography animation, immobilized metal affinity chromatography

Id: M_wSvG4NcOk

Length: 15min 28sec (928 seconds)

Published: Mon Jan 18 2016