Sei sulla pagina 1di 2

PROTEIN ASSAY USING THE BRADFORD METHOD

AUTHORS
Anjeli Mae Aldueza, Fredrick Romulus Altea, Vanessa Amistad, Jose Ang Jr., Alyssa Eireen Arcega Group 1, 2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
The Bradford assay is a rapid and accurate method for the estimation of protein concentration. The technique is simpler, faster than the Lowry method, and is subject to less interference. The objective of this part of the experiment is to quantitatively determine protein concentration in a given sample through Bradford assay. A series of test tubes were prepared with the first test tube containing the 1.5mL of distilled water and the last test tube containing 1.5mL of bovine serum albumin standard. The Bradford reagent of 1.5mL was added to each tube and mixed. It was stood for 5 minutes and the absorbance at 595nm was determined using the spectrophotometer. The albumin standard curve was the constructed by plotting A595 against concentration and the concentration of proteins was determined.

INTRODUCTION
The Bradford assay is a faster, involves fewer mixing steps, does not require heating, and gives a more 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. The response is also protein dependent, and varies with the composition of the protein. These limitations make protein standard solutions necessary. A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons. When, a photon encounters an analyte molecule, 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.

gathered, a series of test tubes were prepared containing the following: Test tube no. 1 2 3 4 5 6 7 8 9 mL standard 0 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 mL H2O 1.50 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05

MATERIALS AND METHODOLOGY


The materials used in the experiment include the Bradford assay, Bovine serum albumin (BSA) standard, test tubes, and a UV-Vis Spectrophotometer. After the materials were

Each test tube was added with 1.5mL of Bradford reagent and mixed well. The group let it stand for 5 minutes. The group then transferred an enough amount of each sample to a series of cuvettes and was placed inside the spectrophotometer. The first cuvette served as the blank while the rest of the cuvettes contained the samples. Each absorbance was read at 595nm. The albumin standard curve was then constructed by plotting A595 against concentration and the concentration of proteins was determined.

RESULTS AND DISCUSSIONS

The Bradford assay is commonly used to determin the total protein concentration of a sample. Using the linear regression method, the slope and y-intercept were determined. Table 1. Data of Bradford Assay Test tube 1 2 3 4 5 6 7 8 9 BSA(mL) 0 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 Water (mL) 1.50 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05 Bradford reagent 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Absorbance 0 0.868 1.012 1.171 1.188 1.620 1.227 1.240 0.911

REFERENCES
Crisostomo,A., Daya M., de Guia R., Farrow F., Gabona M., Liu I.,Pena G.,Pena L., Santiago L.,Santiago M., Sarile A., Torres P., Vargas A., Ysrael M., et al Laboratory Manual in General Biochemistry Quezon City: C & E Publishing Inc. Del Rosario A., et al Isolation and Characterization of Casein Bradford Protein Concentration Assay http://wolfson.huji.ac.il/purification/Proto cols/Bradfordassay

Graph 1. Standard BSA curve 2 1.5 1 0.5 0 0 5 10 Series1