Protein and DNA concentration determination Lab Report Laboratory 1 BTEC 3P93

Tatiana Machado Rodrigues da Cunha Instrutor: Matilda

January 30, 2014

1. Introduction
The determination of the concentration of a protein sample is quite important for any biochemical research that involve proteins studies. This is due the necessary of this information for the characterization and purification of the proteins of enzymes. Currently exists a variety of protein determination methods and each one is suitable for different applications and the appropriate methodology must be chose for the specific problem. For example, determining the total amount of protein concentration is helpful in the fermentation experiments, because the cell growth can be measured by simply centrifuging the aliquot of broth, which the supernatant (proteins) allows a measure of the results of the fermentation. For choose the appropriate method some criteria must be considered, which includes the amount of protein available to assay; the concentration of protein; the specificity of the assay; the presence of chemicals that may interfere with the assay and the ease, and reliability of performing the assay. The quantity of protein is important because in some cases the protein sample is destroyed, so it is needed a big amount of protein for use the method and if have a small quantity of it is recommended to choose a sensitive detection method. As the total of the protein, the concentration of the protein is also essential to consider in choosing an assay. In cases that is necessary a narrow range of protein concentration is needed to do various measurements of dilute solutions of protein to obtain a good response. The specificity refers to the assay only responding to the desired protein and no other component (contaminants) or protein; each protein has its own characteristics that provide different responses and different standard curves for diverse assay techniques. Bovine serum albumin (BSA) is often use for make standard curve of unknown

proteins, although not always is the same response. To make a good standard in a protein assay is necessary the purified preparation of the protein. Usually the proteins is performed in buffered solutions that contains chemical compounds that does not interfere in the protein determination, otherwise detergents and some of other chemicals compounds can affect the results in determining the concentration of the protein. Lastly, the protein assay should be fast and easy to perform with fewer limitations, which should provide to the researcher precision in the results. In the experiments was used three different colorimetric procedures for quantification of proteins, Ultraviolet absorption (UV) spectroscopy, Dye-binding assay (Bradford) and Bicinchoninic acid (BCA) assay. The concentration of protein detected by UV is measured in the 280 nm absorb light. Its advantages is due it does not destroy the protein sample and no need much instrumentation being very rapid. Often proteins have an absorption maxima 200 and 280 due the many amide carbonyls and the aromatic residues (tryptophan, tyrosine and phenylalanine), respectively. In the 200 nm region the absorption is very intense and other organic compounds also absorb at it. While in the absorption at 280 nm the detection is less sensitive and also more flexible since not all proteins have the same amount of aromatic residues. Therefore, the method is not very accurate except if the protein is pure and the molar absorptivity is know. The majority of the proteins at 1 mg/mL (or equivalently 0.1% solution) have an absorbance of between 0.5 and 2.5. With the UV spectroscopy is possible to detect and measure nucleic acid due the aromatic groups in the purine and pyrimidines bases that absorption maxima is 260 nm. It is also possible to have nucleic acids contaminants in the protein sample concentrations, thus with this equation is possible to determining the purity if the protein sample: Concentration (mg/mL) = 1.55 A280 - 0.76 A260

In the method call Bradford the binding of the dye, Coomassie Brilliant Blue G250 to proteins causes a shift in the absorption maxium of the dye from 465nm to 595 nm in acidic solutions. This compound forms strong, noncovalent complexes with proteins via electrostatic interactions with amino groups and carboxyl groups, and by van der Waals forces. It is recommended that a standard curve be made with the assay meanwhile the color response does not maintain linearity over wide range of protein concentration. This technique is rather sensitive and accurate, however the reagent stains cuvettes is somewhat hard to remove. Also is compatible with most of the common buffers and reagents used in these assays. The Bicinchoninic acid (BCA) method for protein determination is one of the most recent protein assays advances. The reaction is similar to the Lowry reaction, excepting that the BCA is used to replace the Folin-Ciocalteu reagent. The protein in alkaline solution can reduced Cu2+ to Cu1+. The BCA molecule chelate to a cuprous ion resulting in an intense purple color that have an absorbance maxium at 560 nm. This assay has a sensitivity similar to the Lowry method and has selectivity for the cuprous. Although it take some time to measure, just after 30 minutes incubating because of the color.

2. Results
Were made 4 procedures to determining the concentration of samples of BSA and IgG with these three protein assays methods using standards solutions of these proteins. In the first procedure the BSA standard solution (1.0 mg/mL in 50 mM phosphate buffer, pH 7,4) was measure in the spectrophotometer with absorption at 280

nm. The absorbance obtained was 0,669 and with the following equation was calculated the absorption coefficient for BSA. Concentration (%) = absorbance / A (1 % solution and 1 cm pathlength) A = absorbance / Concentration A = 0,669/ 0,1 A = 6,69 With it the concentration of the unknown (C) solution of BSA was calculated with the absorption coefficient given and the coefficient above. The absorbance measured of this solution was 0,066. Concentration (mg/mL) = absorbance / A (1 mg/mL and 1 cm pathlength) With the absorption coefficient calculated, A = 6,69: Concentration = 0,066 / 6,69 Concentration = 0,0098 mg/mL With the absorption coefficient given, A= 6,3: Concentration = 0,066 / 6,3 Concentratiom = 0,0104 mg/mL In the second procedure was determined the absorbance of the unknown solutions containing protein and acid nucleic mixture at 260 nm and 280 nm; then was calculated the protein concentration using the equation below. The absorbance obtained was: A260 = 0,512 and A280= 0,499

Concentration (mg/mL) = 1.55 A280 - 0.76 A260 Concentration = 1.55x 0,512 0.76x0,499 Concentration = 0.793 0.379 Concentration = 0.414 mg/mL of protein The procedure third was done the Dye Binding (Bradford) method, which after prepared five dilutions of IgG and BSA standard the absorbance was measured at 595 nm, also was measured the unknown solutions of each protein. The results are show in the following table. [IgG] g/mL 500 g/mL 350 g/mL 250 g/mL 125 g/mL 62,5g/mL Absorbance [BSA] g/mL 0,239 0,172 0,100 0,032 0,008 1000 g/mL 750 g/mL 500 g/mL 250 g/mL 125 g/mL Unknown(A) Absorbance 0,847 0,649 0,458 0,260 0,110 0,127

Unknown(A) 0,097

Table 1. Procedure 3 Bradford: Absorbance of IgG and BSA dilutions. With these results, the standard curve of both protein was constructed and the concentration of the unknown protein was also determined.

Standard curve of IgG - Bradford

0.3 0.25 y = 0.0005x - 0.0307 R = 0.9938

Absorbance (A595)

0.2 0.15 0.1 0.05 0 0 100 200 300 400 500 600

Protein concentration (g/mL)

Chart 1. Standard curve of IgG of the procedure 3, Bradford.

Standard curve of BSA - Bradford

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 200 400 600 800 1000 1200 y = 0.0008x + 0.0331 R = 0.996

Absorbance (A595)

Protein concentration (g/mL)

Chart 2. Standard curve of BSA of the procedure 3, Bradford. For the IgG unknown solution the concentration was: Using the formula y = 0,0005x - 0,0307 , where the y is the absorbance and x the concentration we have: 0,097 = 0,0005x 0,0307 X = 0,097 + 0,0307/ 0,0005

X = 255,4 g/mL And for the BSA unknown solution the concentration was: y = 0,0008x + 0,0331 0,127 = 0,0008x + 0,0331 X = 0,127 - 0,0331/ 0,0008 X = 117,37 g/mL In the last procedure was done the Bicinchoninic Acid (BCA) method that also needed dilutions and measured the absorbance, but at the 562 nm. [IgG] g/mL Absorbance [BSA] g/mL 500 g/mL 350 g/mL 250 g/mL 125 g/mL 62,5g/mL 1,103 0,844 0,594 0,326 0,180 1000 g/mL 750 g/mL 500 g/mL 250 g/mL 125 g/mL Unknown(A) 1,365 1,069 0,745 0,407 0,236 0,399 Absorbance

Unknown(A) 0,737

Table 2. Procedure 4 BCA: Absorbance of IgG and BSA dilutions. With these data, the standard curve of the both proteins was plotted as showing below. The concentration of the unknown proteins also was calculated. For the the IgG, using the formula y = 0,0021x + 0,0592 was: y = 0,0021x + 0,0592 0,737 = 0,0021x + 0,0592

X = 0,737 - 0,0592 / 0,0021 X= 322,76 g/mL

For the BSA, using y = 0,0013x + 0,0843 was: y = 0,0013x + 0,0843 0,399 = 0,0013x + 0,0843 X = 0,399 0,0843 / 0,0013 X = 242,07 g/mL

Standard curve of IgG - BCA

1.2 1 0.8 0.6 0.4 0.2 0 0 100 200 300 400 500 600 y = 0.0021x + 0.0592 R = 0.9962

Absorbance A(A562)

Protein concentration (g/mL)

Chart 3. Standard curve of IgG of the procedure 4, BCA.

Standard curve of BSA- BCA

1.6 1.4 y = 0.0013x + 0.0843 R = 0.9992

Absorbance A(A562)

1.2 1 0.8 0.6 0.4 0.2 0 0 200 400 600 800 1000 1200

Protein concentration (g/mL)

Chart 4. Standard curve of BSA of the procedure 4, BCA.

3. Discussion
As was discuss in the introduction it is known that each protein due its different characteristics both physical and chemical has different responses to each type of method for determining the concentration of those proteins. Thus, the standard curves will differ for each protein for different assays techniques, which applies for this experiment with the IgG and BSA did with the Bradford and the Bicinchoninic Acid (BCA) methods. Analyzing the curves of the Bradford method it shows that the BSA exhibits a strong dye response, with higher absorption at the 595 nm than IgG. One disadvantage of using BSA is that may under-measure the protein content. In the BCA curves was possible to notice a similar absorption for both proteins; compared to other methods this assay is one of the most sensitive, it has less variability than others (i.e., Bradford assay), and it can be used to measure a wide range of protein concentration. Each protein in a sample responds uniquely in a given protein assay. Such protein-toprotein variation refers to differences in the amount of color (absorbance) obtained when the same mass of various proteins is assayed concurrently by the same method.

These differences in color response relate to differences in amino acid sequence, isoelectric point (pI), secondary structure and the presence of certain side chains or prosthetic groups. In the first procedure using the Ultraviolet absorption (UV) spectroscopy, the concentration of the unknown protein was 0,0098 (A=6,69) and 0,0104 (A=6,3) mg/mL, for the Dye-binding assay (Bradford) method the concentration of the unknown protein was 255,4 g/mL to the IgG and 117,37 g/mL to the BSA; and for the BCA method the concentrations was 322,76 g/mL to IgG and 242,07 g/mL to BSA. Although, error in the experiment can be a cause of a lot of factor: the pre-diluted standards are conveniently packaged eliminating wasteful and sharp ampoules, and ensuring protein stability over the shelf life. Pipetting of the reagents and the dye can cause problems such as the inadequate or too much addition of both. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve in the selected protein assay method and is readily available to any laboratory at any time. Therefore, it is advisable to choose a protein standard that is likely to give absorbance values close to those for the protein samples of interest (e.g. if you determine the concentration of an immunoglobulin, use IgG as a standard).

4. Conclusion
With this experiments the students was able to understand the proteins assays and how use each one and how choose the right one for the determining the protein concentration. It is important to highlight that the absorbance is directly proportional to

the amount of protein present in the solution and it can be estimated by comparison with a known protein standard, such as bovine serum albumin (BSA).

5. References
Hayworth, D. (n.d.). Overview of Protein Assays Methods. Retrieved January 30, 2014, from http://www.piercenet.com/method/overview-protein-

assays#protein-variation Johnson, M. (2013). Protein Quantitation. Retrieved January 30, 2014, from http://www.piercenet.com/method/overview-protein-assays#protein-variation Ninfa, A. J., Ballou, D. P., & Benore, M. (2010). Fundamental laboratory approaches for biochemistry and biotechnology. Hoboken, N.J. : Wiley, 2010.