Affinity chromatography | Introduction and Principle in Hindi

In this video I have discussed about what is Affinity Chromatography in Hindi, what is its principle and lab procedure.
Affinity chromatography technique exploits the property of the biomolecules such as enzymes to bind specifically to the substrate or any functional group
Examples:
1. Enzyme binding to the substrate.
2. Antibody with specific Antigen.
3. Hormone binding to receptor.
First we choose the matrix according to our sample, after that the activation of the matrix is one of the essential steps in order to attach the ligand to the matrix. (Sorry I forgot to mention it in video ) It is generally carried out by the cyanogen bromide. The matrix is reacted with the cyanogen bromide (CNBr) at pH 11 and 20° C temperature. One activation is done, through coupling reaction the ligand will be added.
Loading the sample:
Once the column is prepared, the sample needs to be loaded. Before loading the sample, we first equilibrate the column with the buffer so to make the pH and ionic strength uniform throughout the column.
The sample is loaded in the ratio of 1:10. The unbound sample which does not have an affinity for the ligand will elute out first. The molecules of interest which have high affinity for the ligand would be retarded.
Elution of the sample:
To elute the sample, we use two type of method. One is specific, and the other is non-specific.
The non-specific method includes the change in pH and ionic strength. Change in the pH would reduce the affinity of the molecules to the ligand and thus elute out. It is called pH shift elution. Same story for the increase in the ionic strength. These changes can be brought in the column by using a buffer.
The specific method includes the addition of the macromolecules which can bind to the ligand with higher affinity than the molecule of interest (macromolecule such as protein). Thus, the molecules of interest would be replaced and elute out. One can also add such macromolecules which have higher affinity for the protein or molecules of interest than the ligand. So, the proteins would elute out with the macromolecules leaving the unbound ligand in the column.
There are some different modification to the affinity chromatography. If antibodies or antigens are used as ligands, then it is known as immunoaffinity chromatography. Certain proteins have an affinity for metal ions. In such cases, metal ions such as Cu2+, Ni2+, Zn, Mg, etc. are attached to the matrix, and it is known as Immobilized metal ion affinity chromatography.
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...
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