Welcome to Scribd. Sign in or start your free trial to enjoy unlimited e-books, audiobooks & documents.Find out more
Download
Standard view
Full view
of .
Look up keyword
Like this
27Activity
0 of .
Results for:
No results containing your search query
P. 1
Protein Assay by the Bradford Method

Protein Assay by the Bradford Method

Ratings: (0)|Views: 13,581|Likes:
Published by Michelle

More info:

Published by: Michelle on Jan 08, 2011
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as DOC, PDF, TXT or read online from Scribd
See more
See less

08/19/2013

pdf

text

original

 
--------------BIOCHEMISTRY--------------Biochemistry Laboratory – CH600 (2008-2009) Experiment 3
Protein Assay by the Bradford Method
*Michelle Dy Sim, Gellina Ann Ram Suderio, Jonnah Kristina Chua Teope
 Department of Biology, 3Biology-6, College of ScienceUniversity of Santo Tomas, España 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 unknownconcentration, 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 106.117µg/mL
 
and 88.335µg/mL. The average concentration is 97.226µg/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 sampleand then carries out amino acid analysis on the hydrolyzate. But, this method is time-consuming. 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 tointerference from common buffer substance and is very sensitive and rapid if you use elevatedtemperatures, but, the reaction does not go to completion when performed at room temperature anddilution is often necessary for concentrated protein samples.The Bradford protein assay is aspectroscopicanalytical procedure used to measure theconcentration 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 mixing 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 composition of the protein. These limitationsmake 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. When, 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.The objective of this experiment is to determine the concentration of the unknown proteinsolution and to draw the standard curve by plotting the 595 nm (A
595
) against a reagent blank.
 
II. Methodology
The class was divided into two teams. Team A comprised of groups 1 to 5. Team B comprised of groups 6 to 9. The members of this group were assigned to perform only test tube numbers 3 and 4.Standards were prepared. For test tubes number three and four, 0.3mL and 0.4mL of the BSAstock solution were used. Then, distilled water was added to each tube to bring the volume to 1mL. Toeach of these test tubes, 5mL of Bradford reagent was added.
Table 1. The volume (mL) of each test tube, BSA stock Solution (mL), distilled water (mL), Bradford reagent(mL)
Test Tube #34BSA Stock, mL
0.30.4
Distilled Water, mL
0.70.6
Bradford Reagent, mL
55For the unknown protein solution, 1mL was used. Then, 5mL of the Bradford reagent was added.The spectrophotometer was then zeroed using a test tube 1 as the reagent blank. After fiveminutes, but before one hour, the absorbance of the standards and the unknown protein solution wereread at 595 nm (A
595
). A standard curve was drawn by plotting the A
595
versus the concentration of BSA.
III. Results and DiscussionA. Results
Table 2. The volume (mL) of the BSA stock solution, distilled water, Bradford reagent, the Concentration and the Absorbance of the 1
st
to the 6
th
test tube
Test Tube #123456BSA Stock, mL
00.20.30.40.50.6
Distilled Water ,mL
10.80.70.60.50.4
Bradford Reagent, mL
555555
Concentration µg/mL
0406080100120
Absorbance
-0.00020.14040.20990.26570.31170.3632
Table 3. The volume (mL) of the BSA stock solution, distilled water, Bradford reagent, the Concentration and the Absorbance of the 7
th
, 8
th
and the2 tubes containing the unknown

Activity (27)

You've already reviewed this. Edit your review.
1 hundred reads
1 thousand reads
kedarsharma141 liked this
Richard Bunker liked this
Artikah Shahrin liked this
Brianna Losty liked this
iremuzunsoy liked this
An Le liked this
billy_david_1 liked this
Jhemay Aniceto liked this

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->