Biochemical methods 2.docx

Separating molecules according to charge or polarity. Ion exchange chromatography Comprises of mobile phase and stationary phase. Mobile phase runs over the stationary phase. This allows to separate molecules according to affinity. Stationary phase is comprised of tiny beads with charged groups....

Separating molecules according to charge or polarity. Ion exchange chromatography Comprises of mobile phase and stationary phase. Mobile phase runs over the stationary phase. This allows to separate molecules according to affinity. Stationary phase is comprised of tiny beads with charged groups. Can be + or – Anion- negatively charged molecule. Cation- positively charged molecule. Therefore, anion exchange resin has positively charged groups on the beads so it binds to anions And cation exchange resin has negatively charged groups on the beads, so it binds to cations. Strong exchangers- always ionised. Weak exchangers- only ionised over narrow pH ranges. (better for proteins) Cation exchange if positively charged molecules are added, it binds to negatively charged groups on the beads and moves slowly down the column. if negatively charged molecules are added, it quickly moves down the column as it does not bind to anything. Anion exchange if positively charged molecules are added, it will not bind to positively charged groups on the beads and moves quickly down the column. if negatively charged molecules are added, it moves slowly down the column as it binds to positively charged beads. Used for peptides, nucleotides, DNA and proteins- generally used as purification technique. Net charge of a molecule Many molecules contain more than one charged group. Net charge is the overall charge on the molecule when you add all the individual charged together. Eg. A positive charge and two negative charges adds up to give a negative charge. *NET CHARGES CHANGES AT DIFFERENT PH* This is due to different pKa for the COOH and NH2 groups at different pH’s. To get the molecules that are stuck to the beads to flow out of the column, one way is to change the pH of the buffer, so the molecules are no longer attracted to the stationary phase and flow out. Alternatively, excess of salt can be added. If enough NaCl is added, Na wilk compete with the binding to negatively charge beads, this knocks the amino acids off and it flows down the column. Isoelectric pH pH at which the net charge of a molecule is zero. Different amino acids have this isoelectric pH at different pH’s e.g. aspartate has net charge of 0 at around pH 3.5 whereas lysine has net charge of 0 at around pH 8.5. For a molecule to bind to anion exchange resin, pH needs to be greater than molecules Pi (isoelectric PH). For a molecule to bind to cation exchange resin, pH needs to be lower than molecules Pi (isoelectric PH). When proteins are separated, initially the positive/ negative molecules will flow out of the column, but pH gradient can be used to elute them one at a time. Advantages- straightforward good resolution quick Disadvantages- only for charged molecules. some molecules don’t like the ph or NaCl to be varied. Isoelectric focussing Form of electrophoresis that separates according to charge. A PH gradient is established in the gel SDS-PAGE and isoelectric focussing can be done together to make a effective separation according to size and charge. Reverse phase chromatography Separating molecules according to polarity. Polar molecules contain electronegative atoms, hydrogen bonding groups or charged groups. Eg O-H and N-H Reverse-phase media Silica support in centre and hydrophobic phase around. Stationary phase- hydrophobic beads Mobile phase- polar liquid that gets added e.g. polar solvents. If hydrophobic molecules are added, it sticks to the beads and enters stationary phase and moves down the column slowly. If polar molecules are added, they don’t stick to the beads so in mobile phase and moves down the column quickly. Mobile phase is a mixture of water, methanol and acetonitrile. By altering the ratio of these components, you can make a gradient of the mobile phase more hydrophobic. Reverse phase chromatography is typically run with HPLC machine. First peak is the most polar compound, as you move on it gets more hydrophobic. Can be used for peptides, lipids and small molecules eg drugs Advantages- Flexibility good resolution Disadvantages- not good for molecules with low solubility in water. TLC Takes place on a TLC plate. A pencil line is drawn at the bottom, the samples are spotted on and the plate is put into a solvent (level so solvent needs to be below the line) Stationary phase- TLC plate Mobile phase- solvent TLC plate is made of thin layer of silica gel on glass plate. TLC plate is polar. Solvents are non-polar. Hydrophobic molecules move up the plate with the solvent. Polar molecules stick to the plate and doesn’t move much. Used to visualise lipids on a TLC plate. Exposed to iodine fumes which reacts with double bonds in fatty acids and turn yellow/brown. Rf value can be measured from the plate. Distance moved by sample/ distance moved by solvent. Used for amino acids, lipids etc. Advantages: Can visualise components in a sample. Different solvents can be used to adjust separations. Disadvantages: Small samples only Not good for proteins as it denatures and stick to silica. Separating molecules according to affinity/specificity Affinity chromatography Stationary phase- affinity beads Mobile phase- buffer The beads have something attached to them that the protein of interest binds to well. When mixture of proteins is added, the protein of interest binds to the ligand attached to the beads. The rest of proteins flow through the column. In order to elute the proteins attached to the beads, excess of free ligands is added and competes with the beads and knock them off the beads. The graph has time at the x axis and absorbance at 280nm at y axis (proteins absorbs at 280nm) The first peak is the non-specific proteins coming straight out of the column. The second peak is when excess of affinity ligand is added. Advantages: Easy Quick Excellent purification Disadvantages: Not possible for all proteins Western blotting Used to visualise a specific protein within a mixture of proteins. Run a SDS-PAGE (separating proteins according to size) The gel is stacked in nitrocellulose or PCDF membrane. On either side, filter paper or sponges are placed. Electric field is applied across. All the proteins are negatively charged due to SDS-PAGE. All the proteins transfer onto the membrane from the gel. How to visualise the proteins? Blocking = the membrane loves to bind to proteins so need to block all the remaining sites where proteins haven’t bound yet- membrane is washed with BSA Primary antibody= need an antibody specific for your proteins of interest. Place the membrane of solution containing primary antibodies. Wash thoroughly which washes off the non-specific antibodies. Leaving antibody bound only where it is bound to the specific protein of interest. Secondary antibody= second antibody that recognises the first antibody. It recognises the constant region of the first antibody. Enzyme on second antibody such as alkaline phosphatases for detection. Place the membrane in solution containing 2nd antibody. Wash again to remove non-specific bound antibody. Leaving secondary antibody bound to only primary antibody. Developing= add substrate to enzyme attached to secondary antibody. Bands shows up where protein of interest is located. Phosphatases enzyme produces purple bands. Horseradish peroxidase produces light- need to expose to film to see the bands. Advantages Specificity Sensitive Disadvantages Not possible to extract protein, only used to visualise. Time-consuming. ELISA – enzyme linked immunosorbent assay. Use of antibodies to detect and quantify specific proteins within a complex mixture in solution. Carried out in a 9x6 plate. Indirect ELISA- used for quantitating amount of antibody in a sample. Each well of the plate is coated with an antigen. Blocked with a nonspecific protein. Sample is added. If the sample contains antibodies to the antigen, they bond together. Wells are washed. Add enzyme linked 2nd antibody. Wash again Add enzyme substrate. Turns yellow if enzyme bound. Measure absorbance. Used for HIV test. Sandwich ELISA- used for quantitating amount of an antigen in a sample. Each well of the plate is coated with an antibody. Blocked with a nonspecific protein. Sample is added. If the sample contains antigens to the antibodies, it binds to the antibodies. Wash Add different antibody that’s conjugated to an enzyme. Wash Add enzyme substrate. Turns yellow if antigen present. Measure absorbance Advantages Specificity Sensitive Disadvantages Time-consuming