biochemical methods.docx

UV/visible Spectroscopy Spectroscopy is the study between matter and radiated energy. It can be used to: Identify compounds. Determine molecular structure. Quantify substances. Many compounds absorb UV. UV energy is like bonding in organic molecules. The UV absorbed promotes electrons to higher en...

UV/visible Spectroscopy Spectroscopy is the study between matter and radiated energy. It can be used to: Identify compounds. Determine molecular structure. Quantify substances. Many compounds absorb UV. UV energy is like bonding in organic molecules. The UV absorbed promotes electrons to higher energy level as they get excited. Spectrophotometry is used to detect UV light. 2 kinds of measurements can be: Transmittance- how much light passes through the substance. Calculated as % to see how much light shone through. Absorbance- the amount of light absorbed by the substance. Equation= log10(I0/I) ABSORBANCE DOESNOT HAVE UNITS High absorbance= not much light is going through the sample as its getting absorbed instead. Factors effecting absorbance. Beer’s law- light absorption proportional to the number of absorbing molecules (conc) Lambert’s law- light absorption proportional to path length (width of sample the light is passed through) = Beer-lamberts law DNA absorbs UV light at 260nm. Peptide bond in proteins absorb UV at 190nm. Tryptophan and tyrosine amino acid side chain absorb around 280/274nm. Prosthetic groups Haem (400nm) NADH (340nm) Problems with UV spectroscopy Contamination Light scattering Temperature effect Ph effect Degradation Solvent (weather the solvent the sample is in is absorbing the UV instead such as the plastic cuvette, the plastic tends to absorb a lot of UV light instead of the sample inside). Fluorescence spectroscopy Occurs when a light is shone at a sample at certain wavelength which then excites the electrons to higher energy state then they lose energy due to heat or movement. This causes the electrons to drop back down to ground state, it emits a lower wavelength light therefore higher in energy. This can be measured in spectra. GFP is a protein that’s naturally fluorescent and discovered in a jelly fish and used the most these days in biotechnology. Features of fluorescent molecules Highly conjugated system with double and single bonds Many rings structure. Electron donating (OH and NH2) and attracting groups (=O) Rigid and planar. Difference in shifts in the spectra is due to stoke shift. Stoke shift is the difference between excitation and emission wavelengths. The larger the stoke shift, the more sensitive the assay. Fluorescence spectrophotometers Has a light source A prism to split the light source into different wavelengths. Slits to enable to pick the exact wavelength to use. The light is shone onto the sample. A second set of slits are there to measure the wavelength. The emission light is measured at right angles. *Where this differs is that in absorbance spectroscopy, the emissions light is measured straight through and in fluorescence spectroscopy, the emission light is measured at right angle to avoid any inference. * Fluorescence has advantages over UV/ visible absorbance. Enhanced sensitivity since the background is 0. Increased specificity but fluorescent can be effected due to interference with energy transfer and not all compounds are fluorescent. *Most amino acids such as tryptophan are fluorescent. This can be useful as it can be used to study confirmational changes etc. *Alternatively, protein can be labelled with fluorescent dye. Separating and analysing molecules. DNA, proteins, RNA, hormones drugs etc can be separated for research or other purposes. Size- separate large from small Charge/polarity- sperate with opposite charges, separate highly or less charged or separate polar from hydrophobic. Affinity/specificity- separate according to specific interaction. Methods to separate molecules by size: Size exclusion chromatography Also called gel filtration chromatography or gel permeation chromatography Comprise of mobile and stationary phase. Most chromatography techniques use a column format. A column filled with stationary phase. Flowing through the column is mobile phase. Mixture is added at the top and mobile phase is also added on top. As the sample runs through the column, separation occurs and are collected when it comes out of the bottom of the column. Matrix= material packed into the column (stationary phase Eluant= what comes out of the column Fraction= separate tubes eluant is collected in Resolution= degree of separation of different molecules Matrix is tiny little beads made of cross-linked polymers. The beads create a mesh like porous structure so small molecules can fit through but large cant and excluded. Stationary phase is space inside the beads. Small molecules enter the beads and slowly move down the column. Takes longer to come out of column. Mobile phase is space outside the beads. Large molecules cannot enter so stay outside the beads and moves down quickly with the flow of water. Emerges first out of the column. Dialysis Utilises a semi-permeable membrane, only small molecules can pass through the pores and large cannot. Applications to dialysis De-salting Buffer exchange Advantages: Cheap and easy Disadvantages: Slow Can cause proteins to precipitate out of solution. Sample can get diluted. Sample may stick to membrane. Alternative to dialysis is ultrafiltration. Theory of electrophoresis Basic theory is if charged molecules placed in a field of electrical field will move. The speed at which the molecule moves depends on: Strength of electrical field Net charge of molecule The frictional coefficient Electrophoresis carried out in gels such as agarose. The gels form a mesh structure which acts as a molecular sieve therefore small molecules move easily through, but large molecules move slowly through mesh. The concentration of agarose can be altered to choose the best concentration to allows specific molecules you are interested in to pass through. Gradient gels can also be made e.g. 20% agarose at bottom and 5% at the top- this increases resolution. Can be used to: Check purity of sample Visual different number of molecules in a sample Can be used on DNA, RNA and proteins. DNA agarose gel electrophoresis DNA is negatively charged. DNA fragments are large, so agarose gel is used as it has larger pores. Agarose powder is mixed with buffer to make a liquid. It is then poured into a mould and add a cone (used to form wells) Once gel is set, place it in a tank with buffer and remove the cone. Mix DNA sample with loading dye (contains glycerol which is dense and causes the samples to sink into the wells) and add them to the wells. Electrical field is turned on and dye moved down the gel. One of the wells contains DNA ladder, sample containing series of DNA fragments of known size. Use UV light to visualise DNA bands of fragments of DNA. Electrophoresis of proteins using SDS-PAGE Proteins have a wide range of charges and shapes- these effects the velocity and migration of a protein. To overcome this, we use SDS-Poly-acrylamide Gel Electrophoresis. SDS= sodium dodecyl sulphate (detergent)- it denatures the protein and disrupts secondary and tertiary structure. SDS is also negatively charged so large number of SDS bind to proteins therefore proteins have large net negative charge. Reducing agent is also added to break disulphide bonds. SDS-PAGE is run vertically, and gel is much thinner. Gel is prepared as a liquid and poured into a mould. A comb is added to make the wells. After its set, take the comb out and assemble it in a tank. Protein sample is mixed with Laemmli sample buffer- contains glycerol to help sample to sink into wells. Bromophenol blue is used as a dye to help see the sample to analyse the sample. Sample is loaded into the wells and voltage is applied. To visualise the result, stain such as Coomassie Blue is used. Silver stain can be used which dyes the proteins black/brown but its more expensive and its more sensitive than Coomassie Blue