AbstractThe Bradford assay is quantitative analytical process thatis used to measure the concentration of proteins within a solution. I hadconducted this procedure in order to find the concentration of protein in twounknown protein solutions. 8 BSA solutions were prepared with concentrationsranging from 0.2 mg/ml to 1.0 mg/ml including the two unknown samples. A spectrophotometer,which was set to 595 nm was then used to take the absorbances of the proteinsolutions along with the two unknown protein samples.

This was then used todraw a standard curve of absorbance against concentration. The mean absorbanceobtained  for the unknown proteinconcentration were 0.581 and 0.281The graph was then used to find theconcentrations of the unknown protein sample and the concentration for unknownsample A was 0.

73 mg/ml and for unknown B was 0.32 mg/ml.      IntroductionThere are different types of protein assays which are usedin finding the concentration of proteins, but not all of them produce accurateresults (Sim, Suderio & Teope, 2008). The method which is the only method to produce accurate and reliableresults is by taking a portion of a protein assay sample and acid hydrolysingit, after which you then carry out amino acid analysis on the hydrolyzate. Asmuch as this method is an accurate one, it is also very time consuming which iswhy it is not used very often.The assay which are used mostly in analysing the proteinconcentration are the Folin-Lowry assay, Bicinchoninic Acid assay, the UVabsorbance and the Bradford assay. The Lowry assay works by complexing copperwith nitrogen in the protein and the complexed tyrosine and tryptophan is thenreacted with Folin-Ciocalteu phenol reagent.

This then gives an intense,blue-green colour, which absorbs at 650-750 nm (Cartwright, 2017).With the BCA it works in a similar way to the Lowry assay,as the protein is complexed with copper ions. The only difference is that thecopper bound protein binds to BCA  togive a deep purple colour.

The purple colour is proportional to the amount ofprotein present in the solution. The UV Absorbance oversees the absorbance ofamino acids, such as tyrosine and tryptophan. The advantage of this process isthat it is fast with samples that can be retrieved , but often contaminated bybuffers, biological materials and salts (Sim, Suderio & Teope, 2008).The Bradford assay is one of the most popular proceduresused in measuring protein concentration in a protein solution (Cheng, Wei, Sun,Tian & Zheng, 2016). One of the great things about the Bradford proteinassay is that it has an easy protocol to follow, although some ingredients inprotein drugs mostly detergents often disrupt the Bradford assay even at lowconcentrations.The good thing is that there are processes which can be doneto avoid things such as sample errors within the assay, and the best way toachieve this is by assaying the protein after it has been diluted in severalfold in a compatible buffer (Cheng, Wei, Sun, Tian & Zheng, 2016). This isbecause this process will decrease the number of interfering substance withinthe sample to a lower level where it no longer disrupts the assay.

The Bradford assay involves the use of a Coomassie dye isrequired, as this what the protein binds to and while under acidic conditions, areaction occurs which changes the dye from a brownish colour to blue. TheBradford method measures the presence of standard amino acids residues suchas  arginine, lysine as well as histidine(He, 2011).The Bradford assay requires the need of a spectrophotometerto measure the amount of light that the protein sample absorbs. Thespectrophotometer works by beaming light through the sample at a specificwavelength and it then measures the light intensity reaching a detector. Thelight contains a stream of photons, which are then absorbed by the analytewhich is being analysed in the spectrophotometer.

(Sim, Suderio & Teope,2008).The aim of the practical was to determine the concentrationof protein in two cell extracts using the Bradford protein assay.   Methods andMaterialsThe materials which were used during the Bradford assaypractical can be found in pages 46-48 of the Level 4 Practical andTransferrable Skills Booklet.

The methods that were used in the Bradfordprotein assay can also be found in pages 47-48 of the Level 4 Practical andTransferrable Skills Booklet. During the practical the incubation time wasadjusted from 30 minutes to 15 minutes as to conserve time during thepractical.Results volume of stock BSA in mg/ml Volume of distilled water in µL Final concentration of BSA in mg/ml Amount of BSA in Eppendorf in mg 0 1000 0 0 200 800 0.2 40 400 600 0.4 160 600 400 0.

6 360 800 200 0.8 640 1000 0 1.0 1000 Table 1: Showing the volume of BSAstock, distilled water and final concentrations of BSA stock solution.

In table 1 the final concentration of BSA and the amount ofBSA in the Eppendorf was calculated using the equation for dilution which is C1V1 = C2 V2 which was rearranged to find thefinal concentration.Example of calculationC2= C1 V1/V2 V1=200µL, C1= 1mg/ml, V2= 1000µL1mg/ml x 200µL/1000µL = 0.2 mg/ml  Volume of stock BSA in mg/ml Absorbance replicate 1 Absorbance replicate 2 Mean absorbance 0 0.000 0.

000 0.000 200 0.185 0.163 0.174 400 0.351 0.361 0.359 600 0.

494 0.487 0.491 800 0.

638 0.602 0.620 1000 0.793 0.817 0.

805 Unknown A 0.582 0.579 0.581 Unknown B 0.285 0.277 0.281 Table 2: Showing the first and secondabsorbances of the BSA stock solutions with the mean absorbance at 595 nm.

 Figure 1: Standardcurve for Bradford assay using BSA as the standard protein within a range of concentrationmeasured at 595 nm. DiscussionFrom the set of results obtained from the Bradford proteinsassay, we can see that there is a correlation between the final concentrationof the BSA and the mean absorbance. We see from table 2 in the results sectionthat as the final concentration increases the mean absorbance also increasewhich show a direct correlation between the two sets of data.This is because as the concentration of a solution isincreased, there are more molecules that are present for the light to hit whenit passes through a solution (Kerner, n.d.). So therefore, as the concentrationof the solution increases the more molecules that are present in the solutionmeaning that more light is blocked. It is for this reason that the solution isdarker because it is more concentrated and less light can get through it.

When comparing my results to the literature, it is shown by Provost(2015), that samples should always be run on triplicates, although we used toduplicate the results still correlate with some literature values. For example,when I compared my results with the results of Sim et al (2008), and theresults of Cheng et al (2016), the same pattern of correlation is shown evenwith the fact that they had used triplicates showing that the experiment that Ihad conducted went well.Looking at the results that I had obtained from the Bradfordassay, I can say that there were not any problems associated with thepractical.

Some of the things that I would adjust or change if I were to do thepractical again would be the amount of times I repeated the absorbances andtake a third repeat making it a triplicate.Another change that I would make to this practical if I wereto conduct it again would be rinsing the curvette each time I take a readingfrom the spectrophotometer. This is because I feel that it could improve theresults that I had obtained far more better, although I did start from a lowerconcentration and worked my way up it would have been if I rinsed with the IMSsolution each time I took a reading.    Conclusion through this practical I was able to find the concentrationof the two unknown protein solution by plotting a standard curve of absorbanceagainst concentration. Using the standard curve graph the unknown proteinsolution A had the concentration of 0.73 mg/ml while the unknown proteinsolution B had a concentration of 0.32 mg/ml.we can then see that the Bradford assay tells us that theabsorbance of the BSA is directly proportional to the concentration of the BSA.This is because the absorbance increases as the concentration increases.

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