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2017CHEMISTRYEXAM(NHT) 22
SECTION B – Question 4–continued
Question 4 (6marks)
TheBradfordproteinassayisaspectroscopicmethodofdeterminingtheconcentrationofproteins
inasolution.
TheBradfordproteinassayusesacidiedCoomassieBluedye(G-250),whichchangesfrombrown
tobrilliantbluewhenitbindstoproteinsinasolution.Theintensityofthebluecolourofthedye
increasesastheconcentrationofproteinsinthesolutionincreases.
TheabsorptionspectrausedfortheBradfordproteinassayareshowninthegraphbelow.
400 450 500 550
wavelength (nm)
600 650
465 nm 595 nm520 nm 650 nm
0.8
0.7
0.6
0.5
0.4
absorbance
0.3
0.2
0.1
0.0
Coomassie Blue dye
Coomassie Blue dye
(bound to protein)
Key
a. NamethetypeofspectroscopyusedintheBradfordproteinassay. 1mark
b. Whatwavelengthwouldbemostappropriatetodeterminetheconcentrationofproteinsina
solution?Justifyyouranswer. 2marks

23 2017 CHEMISTRY EXAM (NHT)
SECTION B – continued
TURN OVER
c. The Bradford protein assay was used to determine the concentration of proteins in a liver cell
extract.
• Equalvolumesofstandardsolutionsofproteinswereprepared.Thesamevolumeofdye
was added to each one.
• Thespectrometerwasadjustedtoreadzeroabsorbanceforthe0µg mL–1 standard.
• Theabsorbanceofeachofthestandardsolutionswasmeasuredandthedatawasplottedon
a graph.
 Thegraphbelowshowsthecalibrationcurveforabsorbanceasafunctionoftheconcentration
of proteins.
0.90
0.80
0.70
0.60
0.50
absorbance
0.40
0.30
0.20
0.10
0.00 1000 200 300 400
concentration of proteins (µg mL
–1
)
500 600 700 800
 Priortoitsabsorbancebeingmeasured,1.50mLofthelivercellextractwasmadeupto
10.00mLwithabuffersolution.Usingthesameequipmentwiththesamesettings,the
absorbanceofthedilutedsampleofthelivercellextractwasmeasuredas0.28
i. What is the concentration of proteins in the diluted sample of the liver cell extract? 1 mark
ii. Calculatetheconcentration,inmgmL–1,ofproteinsintheundilutedsampleoftheliver
cell extract. 2 marks