L5 Biochemical Assay PDF

SIJ1005 : Principles of Biochemical Techniques Biochemical Assays  An assay determines the concentration of a given substance in a sample A simple assay direct assay is to weigh the amount of a particular compound or class of compounds in a given sample This is called a gravimetric deter...

SIJ1005 : Principles of Biochemical Techniques Biochemical Assays  An assay determines the concentration of a given substance in a sample A simple assay direct assay is to weigh the amount of a particular compound or class of compounds in a given sample This is called a gravimetric determination In practice, however compounds usually cannot be assayed so directly In biological sample the compound of interest is usually part of a comples mixture of biomolecules >> make it impossible to simply weigh a particular compound separately Thus, indirect assay procedures are used in biochemical analyses This type of assay involves; Select a chemical reaction specific for the compound of interest, i.e. only the compound of interest will participate in the chemical reaction forming the basis of the assay Reacting known amounts of that compound with the appropriate reagents to produce a product that can be measured simply and accurately (standards) Measuring the response produced by a known volume of sample using the identical procedure to that used for the standards. By comparing the absorbance of a known volume of a sample with that given by the standards, the amount of that particular compound in a given volume of a sample can be calculated  The absorbance of the coloured product can be measured Relative to water >>to give an absolute absorbance value Relative to a reference solution (reagent blank) >>to give a relative absorbance Most assay procedures require measurement against a reagent blank The reagent blank contains zero amount of the compound being assayed >>The sample volume is replaced by solvent alone (eg water or buffer) and all steps in the assay are followed The blank is used to set the spectrrophotmeter to zero absorbance, so the standard curve must pass through zero The purpose of the reagent blank is to correct for any contribution to total absorbance made by the reagents used in the colour development The absorbance measured relative to this reagent blank is due to only the reaction the compound of interest present in the reaction mix Nature of standard curves Standard curves are usually linear (Beer’s Lambert Law states that A=kcl) Desired properties of an assay High sensitivity : the assays hould be able to detect very small amounts of the compound of interest >> minimizing the amount of a sample used for the determination Specificity : the reaction should be specific for the compound of interest, i.e. un affected by the presence of other substance Stability : the colour development should be stable enough for absorbance readings to be taken accurately and coveniently Proportionality : the colour change produced should be propotional to the amount of a compound present in the sample Steps involved in performing biochemical assay Choose the assay Choose the standards Decide the range appropriate for each assay Let say >> amount of protein that fall within the suggested range will give a reasonable response in the assay and a substantially linear standard curve This range will be characteristic of the sensitivity of the assay you choose. Thus need to decide How many points will have on the standard curve (5 to 6) The values (amount of protein etc) of the standards sued to construct the standard curve Set up standards A known concentration of standard solution appropriate for the assay Then the calculate the volumes of the standard solution required to give the mounts of the protein  Choose the appropriate volume (s) of unknown solution to assay The aim is to choose a volume of solution of unknown protein solution concentration that will absorb approximately toward the middle, linear section of the standard curve. If the choosen volume results in an amount of unknown substance that is too small>>the absorbance reading may be too low This will affect the reliability of the protein concentration measurements If the volume assayed results in an amount of unknown that is too large >>the absorbance may be outside the range covered by the standards Carry out the assay procedure It is a good procedure to assay the unknowns and standards simultaneously. >>>> any variables associated with instability of the solution, room temperature and instrumental calibration  General points of note when performing assays include: Always prepare standards and unknown at the same time Always mix tubes properly Incubate standards and unknown for the same length of time Measure the ABS of the standard curve and sample and estimate the amount of protein in the unknown sample using a standard curve At the end of incubation period, read absorbance of each solution in spectrophotometer at the specified wavelength When all read plot the standard curve of absorbance vs amount of standard The standard curve can now be used to determine the amount of the protein in the unknown sample The process estimating the quantity that falls between the known data points is interpolation Note that never extrapolate from standard curve Using the amount estimated from the standard curve, the concentration of the unknown solution can be determined >> because it is known what volume of unknown