--------------BIOCHEMISTRY--------------Biochemistry Laboratory CH600 (2008-2009) Experiment 3 Protein Assay by the Bradford Method *Michelle Dy Sim, Gellina Ann Ram

m Suderio, Jonnah Kristina Chua Teope Department of Biology, 3Biology-6, College of ScienceUniversity of Santo Tomas, Espaa Street, Manila 1008 December 12, 2008 Abstract: The Bradford protein assay is aspectroscopicanalytical procedure used to measure the concentration of proteinin a solution. This experiment aims to determine the concentration of the unknown protein solution and to draw the standard curve by plotting the 595nm (A 595 ) against a reagent blank. Standards were prepared by adding 0.3 and 0.4mL of BSA stock solution.Distilled water was added to each of the test tube to bring the volume to 1mL. For the determination of the unknown concentration, 1mL of the unknown protein sample was used. Through the use of the spectrophotometer, the absorbances (innm) for the unknown proteins were determined. A standard curve was drawn by plotting the A 595 versus the BSA concentration.The concentrations of the unknown proteins were solved by using linear regression. The result obtained for the concentration of the unknown for trials 1 and 2 are 106.117g/mL and 88.335g/mL. The average concentration is 97.226g/mL. The averageabsorbance is 0.2929nm. Keywords: Protein Bradford Assay Method Spectrophotometer BSA standard (bovine serum albumin) I. Introduction There is no single protein assay method that yields absolutely accurate results. The only true andaccurate method for determining protein concentration is by acid hydrolyzing a portion of the sam pleand then carries out amino acid analysis on the hydrolyzate. But, this method is timeconsuming. Eachmethod and assay has its own disadvantage and limitations.The most commonly used assays are the Ultraviolet Absorbance, Lowry Assay, BCA assay andthe Bradford Assay. The UV absorbance monitors the absorbance of aromatic amino acids, tyrosine andtryptophan or if the wavelength is lowered, the absorbance of the peptide bond. It is quick, with the

samples that can be recovered. But, it is also highly susceptible to contamination by buffers, biologicalmaterials and salts. The Lowry Assay or enhanced copper since it reduces Cu +2 to Cu +1 , sensitive over awide range and is the most commonly referenced procedure for protein determination but, it also takes aconsiderable amount of time. And the BCA assay or the bicinchoninic acid which is less susceptible toi n t e r f e r e n c e f r o m c o m m o n b u f f e r s u b s t a n c e a n d i s v e r y s e n s i t i v e a n d r a p i d i f y o u u s e e l e v a t e d temperatures, but, the reaction does not go to completion when performed at room temperature and dilution is often necessary for concentrated protein samples.T h e B r a d f o r d p r o t e i n a s s a y i s a spectroscopica n a l y t i c a l p r o c e d u r e u s e d t o m e a s u r e t h e concentration of proteinin a solution. It is dependent on the amino acid composition of the measured protein. It is more efficient than other methods because assay it is faster, involves fewer mix ing steps,does not require heating, and gives a more stable

colorimetric response than other methods.The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives amore stable colorimetric response. Its response is prone to influence from non protein sources and becomes progressively more nonlinear at the high end of its useful protein concentration range. Theresponse is also protein dependent, and varies with the compos ition of the protein. These limitations make protein standard solutions necessary.A spectrophotometer is employed to measure the amount of light that a sample absorbs. Theinstrument operates by passing a beam through a sample and measuring the intensity of light reaching adetector. The beam of light consists of a stream of photons. W hen, a photon encounters an analytemolecule, there is a chance the analyte will absorb the photon. This absorption reduces the number of photon in the beam of light, thereby reducing the intensity of the light beam.T h e o b j e c t i v e o f t h i s e x p e r i m e n t i s t o d e t e r m i n e t h e c o n c e n t r a t i o n o f t h e u n k n o w n p r o t e i n solution and to draw the standard curve by plotting the 595 nm (A 595 ) against a reagent blank The Standard Curve of A 595 VersusThe Concentration of BSA 0, -0.000240, 0.140460, 0.209980, 0.2657100, 0.3117120, 0.3632160, 0.4624200, 0.512997.226, 0.29290.100.10.20.30.40.50.60 5 0 1 0 0 1 5 0 2 0 0 2 5 0 concentration (x) a b s o r b a n c e ( y ) Graph 1. Standard curve for BSA, Absorbance (nm) versus Concentration (g/mL)

B. Discussion The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dyeCoomassie when the previously red form Coomassie reagent changes and stabilizes into Coomassie blue by the binding of protein. During the formation of this complex, two types of bond interaction take place: the red form of coomassie dye first donates its free proton to the ionizable groups on the protein,which causes a disruption of the proteins native state, consequently exposing its hydrophobic pockets.These pockets on the proteins tertiary structure bind non-covalently to the non-polar

region of the dyevia Van der Waals forces, positioning the positive amine group in proximity with the negative charge of t h e d y e . T h e b o n d i s f u r t h e r s t r e n g t h e n e d b y t h e i o n i c i n t e r a c t i o n b e t w e e n t h e t w o . B i n d i n g o f t h e protein stabilizes the blue form of coomassie dye, thus the amount of complex present in solution is a measure for the protein concentration by use of an absorbance reading. Figure 1. Coomassie Brilliant Blue G-250 structure ProteinRed <=> Green <=> Blue <=> Blue-Protein(470 nm) (650 nm) (590 nm) (590 nm)H + H + Figure 2. Equilibrium of Coomassie brilliant blue G-250 The assay is useful since the extinction coefficient of a dye-albumin complex solution is constantover a 10-fold concentration range.

Because the Bradford assay essentially measures the amount of arginine and hydrophobic aminoacid residues, the amino acid composition can alter the concentration -absorbance curve depending onthe percentage of arginine or hydrophobic amino acids in each protein. It is therefore necessary to use astandard (e.g. BSA-- Bovine Serum Albumin) whose protein closely matches the measured protein incompositionT h e d y e r e a g e n t r e a c t s p r i m a r i l y w i t h a r g i n i n e r e s i d u e s a n d l e s s s o w i t h h i s t i d i n e , l y s i n e , tyrosine, tryptophan, and phenylalanine residues. Obviously, the assay is less accurate for basic or acidic p r o t e i n s . T h e Bradford assay is rather sensitive to bovine serum albumin, more so than " a v e r a g e " proteins, by about a factor of two. Immunoglogin G (IgG - gamma globulin) is the preferred proteinstandard. The addition of 1 M NaOH was suggested by Stoscheck (1990) to allow the solubilization of membrane proteins and reduce the protein-to-protein variation in color yield.E r r o r in the experiment can be a cause of a lot of factor: the prediluted s t a n d a r d s a r e conveniently packaged eliminating wasteful and sharp ampoules, and ensuring protein stability over theshelf life. Pipetting of the reagents and the dye can cause problems such as the inadequate or too muchaddition of both.Spectrophotometer should be down to the zero point by the reagent blank since it can be a ver y big factor for error in the experiment. It is also suggested that at least two reagents blank should be performed or one can use a water buffer instead. But, if the spectrophotometer was not zeroed with the blank, take the average of the blank value from the standard and unknown sample values instead.When the absorbance of protein standard and sample is very low, the 1x dye reagent may be coldf r o m 4 0 storage, and then warm the dye reagent to am bient tem perature. If the a b s o r b a n c e o f t h e standard is accept able, but if the absorbances of samples are very low, then the samples may contain a substance that interferes with the reaction, such as detergents or basic solutions. IV. Conclusion Through the experiment, the group was able to solve f or the concentrat ion of t h e u n k n o w n protein solution by using the linear regression method and by plotting the standard curve by absorbanceversus concentration. Using the standard curve, The unknown protein solution had a concentration of 1 0 6 . 1 1 7 g / m L a n d 8 8 . 3 3 5 g / m L . T h e a b s o r b a n c e o b t a i n e d

f o r t h e u n k n o w n a r e 0 . 3 1 3 3 n m a n d 0.2724nm. The average concentration is 97.226g/mL. The average absorbance is 0.2929nm.The Bradford method shows that the absorbance has a direct relationship with the concentrationof the protein sample, meaning if the absorbance is high, the concentration of the sample is also high.