BIOL 541 1st Edition Lecture 4 Outline of Current Lecture Examples of chromatographyCurrent Lecture 1. Thin layer chromatography: It is also referred as absorption chromatography when paper is used as a medium. Glass sheets and metal sheets can be used as a medium. Silica gel which is a hydrophilic agent is placed in a glass tank and covered with a lid. There is also a mixture of organic solvent saturated with water and kept in a beaker. At the bottom, protein is spotted. When the beaker floods the bottom of the tank, mixture goes up due to capillary action. Here the aqueous water acts as the immovable phase and the mixture of solvents is the movable or organic phase. The aqueous compound dissolves protein and the more hydrophilic protein spends more time in the aqueous phase and stays there. The hydrophobic proteins enter the solvent- mobile phase- and move up. There is a difference in distance (partition) between phases and hydrodynamic movement of solvent across the plate. There is also an impeding forcethat is the partition between the phases.Disadvantage: There is a possibility of not doing large scale purification. Further, not all proteins are hydrophilic, hence they not move very far in the medium.2. Reverse phase chromatography: If it is adapted for protein separation, it is called glass column chromatography. The column is filled with glass beads that are hydrophobic. The salt concentration is increased in order to buffer the proteins out of the solution. In otherwords, the protein enters the beads. Decreasing the salt gradient enables the proteinto move with the hydrophilic buffer down the column. Here the retarding force enables distribution of protein.3. Gel chromatography: Differences in the protein size and shape enable protein distribution. Carbohydrate beads are cross linked with variability to create pores. More levels of cross linking decreases the pore diameter. Hydrophilic carbohydrates absorb the buffer and enter the center of the beads, thereby creating a stationery phase. The buffer migrates to the bottom. The repelling force is the hydrodynamic force. Largeproteins can not enter the pores and are stuck in the mobile phase, hence move down the column. The smaller sized proteins enter the pores and hence can not move down. Due to Brownian motion, the small ones move out of one glass bead into another, hence moving down the column in a slow motion. Large proteins and small proteins can be collected in different test tubes. The intermediate sized proteins are partially retarded and come in the middle. Size and shape of the protein is dependent upon the geometry of the pores, hence can be termed as molecular sieving chromatography.4. Charge chromatography: Here the matrix is a cross linked gel. The (-) charge is added to protein by coupling with carboxylic acid or sulphates. Protein sticks to the opposite charge of the matrix. Slowly increasing the pH of the buffer causes pH to reach isoelectric point enabling protein to fall off the column. Example: as P .A has a weaker bond than P.B , A comes out first then B.5. Cation exchange: Since it is very difficult to establish pH gradient, salt gradient is used.Increasing the Sodium concentration in buffer from 0 to 1M causes Sodium to outcompete the protein, as Sodium has a higher (-) charge density. Based on the attraction of protein, Protein A is knocked out first. Then increasing the Sodium concentration again knocks out Protein B.6. Anion exchange: Here amino group is added to enable the protein to come out based on charge gradient and not salt. Here sharper separation is possible based on the true Pi values of the protein.7. Electrophoresis: Here the neutral matrix is the filter paper which is moistened. The sample is added followed by adding charge across the paper. Proteins migrate towards the (-) charge. The more charged proteins move rapidly enabling separation.Here the charge is the electrostatic force and friction is the repelling force. Gel polyacrylamide is used as it is better in analytical abilities than filter paper. Few mgms of proteins are separated by this method.8. Affinity chromatography: There is high affinity between the matrix and protein. Matrix has specific binding sites for different shapes of protein. Protein buffers through except those bound to the matrix. The column is washed to remove the bound proteins. In other words, the salt concentration or the charge concentration is increased toenabling removing of the protein. This method has high specificity and high affinity magnitude of 100’s fold magnification.Enzyme substrate interaction: Either the substrate is attached to matrix or the product is attached to the matrix, such as product inhibition works as feedback inhibition. When co- enzyme (organic molecule) is attached to matrix, modulators can also be attached to matrix.Antibody- antigen: Tight antibodies are added to matrix and gives specific proteins or proteins added to matrix, then given to rabbit.Lectins: These are sugar specific proteins or glycoproteins whereby there is a specific lectin for specific protein. Thereby, one type of protein sticks to lectin, whilst others pass through.Receptors: Hormone is attached to matrix, followed by grinding the cells. These are passed down the column, whereby the receptor sticks to the column.Transcription factors: Here the DNA sequence must be known. The nucleotides are synthesized and attached to matrix. Then the nuclear extract must be passed down the column, so that the transcription factors that recognize the sequence will stay.Factors to consider for protein separation method choice:1. The choice of high capacity low cost method is done first followed by low capacity high cost method.2. This will enable sampling of liters of materials before mgms of materials.3. Always start with precipitation techniques followed by affinity chromatography as the affinity chromatography is expensive.4. Always give preference for gentler techniques as proteins that are used in biological activities can be easily denatured. Only if the sequence is known,