E STIMATION O F P ROTEIN C ONCENTRATION I N E GG A LBUMIN B Y L OWRY M ETHOD

I NTRODUCTION
The Lowry Assay: Protein by Folin Reaction (Lowry et al., 1951) has been the most widely used method to estimate the amount of protein (already in solution or easilysoluble in dilute alkali) in biological samples. First the proteins are pretreated with copper ion in alkali solution, and then the aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic acid present in the Folin Reagent. The end product of this reactionhasabluecolor.Theamountofproteininthesamplecanbeestimatedviareadingthe absorbance (at 750 nm) of the end product of the folin reaction against a standard curve of a selectedproteinsolution(inourcase;BovineSerumAlbumin(BSA)solution). TheLowrymethodreliesontwodifferentreactions. 1. The first reaction is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions. This is called a Biuret chromophore and is commonlystabilizedbytheadditionoftartrate(Gornalletal.,1949). 2. ThesecondreactionisreductionoftheFolinCiocalteureagent(phosphomolybdateand phosphotungstate), primarily by the reduced copperamide bond complex as well as by tyrosineandtryptophanresidues. The Biuret reaction itself is not very sensitive. Using the FolinCiocalteu reagent to detect reduced copper makes the Lowry assay nearly 100 times more sensitive than the Biuret reactionalone.SeveralusefulmodificationsoftheoriginalLowryassayhavebeendevelopedto increase the dynamic range of the assay over a wider protein concentration (Hartree, 1972), to make the assay less sensitive to interference by detergents (Dulley and Grieve, 1975), and to first precipitate the proteins to remove interfering contaminants (Bensadoun and Weinstein, 1976). The Lowry assay is relatively sensitive, but requires more time than other assays and is susceptibletomanyinterferingcompounds.Thefollowingsubstancesare known to interfere with the Lowry assay: detergents, carbohydrates, glycerol, Tricine, EDTA, Tris, potassium compounds, sulfhydryl compounds, disulfide compounds, most phenols, uric acid, guanine, xanthine,magnesium, and calcium. Many of these interfering substances are commonly used in buffers for preparing proteins or in cell extracts. This is one of the major limitationsoftheassay.TheLowryassayisalsosensitivetovariationsinthecontentoftyrosine andtryptophanresidues,atraitsharedwiththeultravioletassayat280nm).Theassayislinear over the range of 1 to 100g protein. The absorbance can be read in the region of 500 to 750 nm, with 660 nm being the most commonly employed. Other wavelengths can also be used, however, and may reduce the effects of contamination (e.g., chlorophyll in plant samples

interferes at 660 nm, but not at 750 nm). Also, if the A660 values are low, sensitivity can be increasedbyrereadingthesamplesat750nm.

M ATERIALS & C HEMICALS

1. 2. 3. 4. 5. 6. 7. 8. 9. CleanGlasswares CentrifugeTubes(10ml) semimicrocuvettes Spectrophotometer(750nm) BovineSerumAlbumin(BSA) FolinandCiocalteusPhenolReagent(2N) Sodiumtartrant:Na2Tartrant.2(H2O) Coppersulphate:CuSO4.5(H2O) Otherchemicals:NaOH,Na2CO3

P REPARING THE S OLUTION

The Lowry Solution is a mixture of the above chemicals, except the Folin Reagent. The Lowry solution should be prepared fresh, at the day of measurement. Though the individual solutions for the Lowry solution can be prepared in advance and then mixed atthedayofmeasurement. SolutionAisadilutealkalisolution.2NFolinandCiocalteusPhenolReagentcontainHCl andH2PO4.

SolutionA:(alkalineSolution)(for100ml) 0.572gmNaOH 2.862gmNa2CO3 SolutionB:(for20ml) 0.285gmCuSO4.5(H2O) SolutionC:(for20ml) 0.571gmNa2Tartrant.2(H2O) LowrySolution:(freshlyprepared,0.7/mlsample) Sol.A+Sol.B+Sol.Cwitharatio(vol:vol)of100:1:1 FolinReagent(instantfresh,0.1ml/sample) 5mlof2NFolinandCiocalteusPhenolReagent+6mlofdoubledistilledwater this solution is light sensitive. So it should be prepared at least 5min of the first sampleincubationandkeptinanambercontainer.

BSAStandardProteinSolution(fresh) Although BSA is a water soluble protein, it takes time to dissolve it completely. So, prepare this stock solution and keep it mixed i.e., for 1 hour before starting the experiment. weigh0.05gmofBSAandaddtoa500mlvolumetricflaskcontainingddlwater. Stir well to dissolve and adjust the volume to 500 ml with ddl water. Final concentrationofthestocksolutionis100mgBSA/L. Preparethedilutionin15mltubes,followingtherecipegivenbelow Vol.ddlwater Vol.ofstockBSAsolution FinalConcentration DilutedSampleno. 10ml 8ml 6ml 4ml 2ml 0ml 0ml 2ml 4ml 6ml 8ml 10ml 0mg/ml 20mg/ml 40mg/ml 60mg/ml 80mg/ml 100mg/ml dil.Std1 dil.Std2 dil.Std3 dil.Std4 dil.Std5 dil.Std6

P ROTOCOL
1. ThestockBSAsolutionwaspreparedanddilutedforthestandardcurve. 2. TheLowrySolutionwaspreparedbymixingSol.A+Sol.B+Sol.Cwitharatio(vol:vol)of 100:1:1 3. thesampleswerevortexedanddilutedwithddlwater 4. 0.5mlofthedilutedsample(orstandard)wastransferredto10mlcentrifugetube 5. 0.7mlofLowrySolutionwasaddedtothesamecentrifugetube 6. Thecentrifugetubeswerecappedandvortexedwelltomix 7. Thetubeswereincubatedfor20minatroomtemp.indark. 8. Atthelast5minoftheincubation,FolinReagentwasprepared 9. After the 20 min incubation, the samples were taken out and 0.1 ml of diluted Folin Reagentwasaddedtoeachtube 10. Thetubeswerecappedandvortexedimmediately 11. Thetubeswereincubatedfor30minatroomtemp.indark 12. Whilewaiting,theUVVISspectrophotometerwaswarmedupandstabilized 13. Afterthe30minincubation,thetubeswerevortexedbriefly 14. Thenthe1.3mlofsamplesweretransferredtosemimicrodisposablecuvettes 15. Absorbancereadingweretakenat750nm 16. Thecalibrationcurveformtheabsorbancereadingsofthestandardswerepreparedand theproteinconcentrationinthesamplesinmgBSA/litwascalculatedformthecurve.

P ROTOCOL S UMMARY
ForStandardBSAsolution: Std.Sampleno. dil.Std1 dil.Std2 dil.Std3 dil.Std4 dil.Std5 dil.Std6 ForEggalbumin(Unknown)solution: DilutionofEggalbuminisdonesimilartothedilutionofBSA Unknown Vol.ofstd Vol.ofLowry Sampleno. dil.sample1 dil.sample2 dil.sample3 dil.sample4 dil.sample5 dil.sample6 transferredtotubes 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml Solutionadded 0.7ml 0.7ml 0.7ml 0.7ml 0.7ml 0.7ml Vol.ofstd transferredtotubes 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml 0.5ml Vol.ofLowry Solutionadded 0.7ml 0.7ml 0.7ml 0.7ml 0.7ml 0.7ml Vol.ofFCreagent added 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml Tocuvettes 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water

Vol.ofFCreagent added 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml

Tocuvettes 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water 1.3ml+distill.water

R EFERENCE
Protein (Lowry) Protocol, Protein Protocol, Ebru Dulekgurgen UIUC04, (Author Unknown)