spectrophotometric determination of protein concentration
DESCRIPTION
Spectrophotometric Determination of Protein ConcentrationTRANSCRIPT
-
Presentation onSpectrophotometric Determination of Protein ConcentrationPresented By
Tasnim Zaman Anika
Std no-1202007
Md.Touhidul Islam
Std no.-1202011
Syed Ahmed Tasnim
Std no-1202025
Date: 20-12-15
-
Presentation outlineObjectivesDiscussion on 3 spectrophotometric methodsApplications
*
-
ObjectivesDetermination of protein concentration using three spectrophotometric methods*
-
Three Spectrophotometric MethodsBicinchoninic acid (BCA, Smith) Method.
Bradford Method.
Direct absorbance measurement*
-
Principle:
The purple color resulting from this method is due to:
A] The nitrogens in peptide bonds in protein, reduce Cu2+ ions to Cu+
(a temperature dependent reaction)
B] Cu+ chelated by two molecules of BCA, to produce a
copper-BCA complex [purple color] with maximum absorption
(max) of 562 nm.*1.Bicinchoninic acid (BCA, Smith) Method:
-
Reaction*
-
*Advantages:Because the peptide backbone is involved in the reaction, the BCA assay is less sensitive to the types of amino acids in the protein.Disadvantages:The presence of reducing agents in your buffer can interfere with the dyeThe reaction takes some time to proceed
-
2.Bradford Method:*a colorimetric assay based on the interaction between Coomassie brilliant blue and the arginine and aromatic residues in your protein-Binding of the dye Coomassie Brilliant Blue G-250 to protein in acidic solution causes a shift in wavelength of maximum absorption (max) of the dye from 465nm to 595nm.
Vapour liquid equilibrium
-
Reaction*Test tubes containing:Bradford reagent alone. (max) of the dye 465nm Test tubes containing: Bradford reagent with protein added.(max) is shifted to 595nm.
-
*Advantages:This assay is quick, and the reagent is not affected by the presence of reducing agentsDisadvantages:If protein doesnt contain a decent number of arginine and aromatic residues, then the dye will not bind to the protein as efficiently, resulting in an underestimation of protein concentration
-
*Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nmIf extinction co-efficient for specific protein is known, the absorbance can be measured in a UV/Vis spectrometerCalculate the concentration of protein using Beers law (A = elc, where l is the path length of the spectrometer)3.Direct absorbance measurement
-
Graphical Representation*Figure : Absorbance Vs Concentration
-
*Advantages:quick and doesnt require any special reagentsDisadvantages:This method relies on having an accurate extinction coefficient, which depends on the number of aromatic residues.If there arent a decent number of aromatic residues, extinction coefficient will be quite low, and a fairly concentrated sample will be needed to get a reasonable absorbance
-
*Applications :Determining the exact quantity of proteins in a solution is very often necessary in the biochemical practiceSpecific protein and its order can be identified by measuring protein concentration
-
Conclusion *All the three methods discussed here are important for different protein analysisQuantitative determination of protein is important
-
THANK YOU ALL
24 Jan 2010Vapour liquid equilibrium*Vapour liquid equilibrium24 Jan 2010*Vapour liquid equilibriumVapour liquid equilibrium24 Jan 2010*Vapour liquid equilibriumVapour liquid equilibrium24 Jan 2010*Vapour liquid equilibriumVapour liquid equilibrium