Protein Quantification of Almond Milk and Egg White Solutions Utilizing the Bradford Assay

Abstract

 The Bradford assay is a popular method for the quantification of proteins due to it relative sensitivity and ease of performance by cuvette. The assay utilizes the Coomassie blue dye, which binds to basic amino acid residues in its anionic form shifting maximum light absorbance to 595nm. Using the known concentration of 1.38mg/ml BSA standard, seven samples were prepared (0,20,40,60,80,100,120ug of BSA) adding appropriate volumes of BSA, DI water (diluted to 100ul), and 5ml of dye to construct a standard curve. Absorbance at 595nm was measured at exactly 5 minutes in each sample. The equation of the line of 0.0111x + 0.0228 (R² = 0.98799) from the BSA standard curve was utilized to determine the protein concentration from two known samples containing 45ul and 65ul of BSA standard and two unknown samples containing 3ul of egg white and 11ul of almond milk. Known protein concentration averages of 1.45mg/ml (45ul) and 1.38mg/ml (65ul) were obtained.  Known concentrations were accurate when compared to the expected value of 1.38mg/ml displaying small percent errors of 0% (65ul sample) and 5.07% (45ul sample). Two unknown protein concentrations of almond milk and egg white presented concentration averages of 2.94mg/ml and 36.76mg/ml. Almond milk (2.94mg/ml) and egg white (36.76mg/ml) samples portrayed concentrations that were much lower when compared to the expected manufacturer values of 4.17mg/ml from Silk Products and 103mg/ml from Golden Eggs. This result indicated that the Bradford assay retained limitations since the Coomassie dye binds specifically to basic residues leading to varying degrees of binding capacity between proteins.

Introduction

 From prokaryotes to eukaryotes, proteins are found in numerous biological systems and are the most abundant class of biomolecules as they represent over 50% of the dry weight of cells (Nouroozi et al., 2015). The quantification of protein concentration in aqueous samples is an important method in biochemistry research for topics including enzymatic studies and nutritional research  (Nouroozi et al., 2015). The Coomassie blue protein assay, commonly known as the Bradford assay, is a widely used spectrophotometric procedure for the quantification of proteins (Gonzalez et al., 2010). The Bradford protein assay is popular due to its relative sensitivity and ease of performance by cuvette (Nouroozi et al., 2015). The dye exists in three forms of anionic (blue), neutral (green; A max 650nm), and cationic (red) (Nouroozi et al., 2015). The mechanism of the assay includes the binding of the anionic form of the dye (Coomassie Brilliant Blue G250) to basic amino acid residues such as tyrosine, histidine, phenylalanine, tryptophan, and especially arginine at an acidic pH (Nouroozi et al., 2015). Under acidic conditions, the dye is in a protonated cationic form (red: Amax=470nm), but as the dye binds to the protein it is converted to a stable anionic (blue) form held together by hydrophobic interactions and ionic bonds (Gonzalez et al., 2010). The formed Coomassie-protein complex subsequently changes max absorbance to 595nm (Gonzalez et al., 2010).

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As more protein binds to the anionic blue dye, the amount of light absorption at 595nm increases leading to a higher protein concentration as described by Beer-lambert’s law (Bio Rad, 1998). BSA (bovine serum albumin) is a protein commonly utilized as a standard due to its stability to increase signal in assays, its low cost, and its ability to lack effect in biochemical reactions (Bio Rad, 1998). The coomassie dye can bind to BSA and the proportional binding of the dye to the protein can be utilized to determine unknown protein quantities (Nouroozi et al., 2015). A BSA standard curve can be constructed utilizing various quantities of BSA standard (20,40,60, 80, 100, 120ug) with the addition of coomassie blue dye; a standard curve of absorbance at 595nm (y) vs. protein quantity (ug) can be constructed and the equation of the line can be utilized to determine unknown protein concentrations (Nouroozi et al., 2015).

The purpose of this experiment is to determine the protein concentrations (mg/ml) for two known samples of 45ul and 65ul BSA standard and two unknown samples of egg white and almond milk utilizing the Bradford assay and a BSA standard curve (Kruger, 1994).

Methods And Materials

Seven samples containing various quantities of BSA standard (1.38mg/ml) were prepared; samples included 0, 20, 40, 60, 80 100, and 120ug of BSA. 7 appropriate BSA volumes (ul) were calculated using the known BSA concentration of 1.38mg/ml and diluted to 100ul utilizing deionized water; the sample containing 100ul of DI water lacking BSA was utilized as a blank. At staggered times, 5ml of coomassie blue dye reagent was added to each test tube (5.1ml total) and vortexed. At exactly 5 minutes, each of the 7 samples was measured for light absorbance at 595nm using a spectrophotometer and the values were utilized to construct a BSA standard curve of absorbance (y) vs. protein quantity (ug) displaying an $R^2$

of 0.98799. The $R^2$

must have been at least 0.98 or else calculated protein quantities would have been skewed and inaccurate. Two known samples (A1,A2,B1,B2) contained 40ul (A1, A2) and 65ul (B1,B2) of BSA standard added to labeled test tubes; 7ul of egg white was added to test tubes C1 and C2 and 11ul of almond milk was added to test tubes D1 and D2. Samples A1-D2 were diluted to 100ul with DI water; at staggered times, 5ml of dye was added to each test tube and absorbance at 595nm was recorded at exactly 5min. The equation of the line from the BSA standard curve was utilized to determine protein quantities from samples A1-D2 and protein concentrations were subsequently calculated.

Results

 The obtained BSA standard curve (1.38ug/ml) was useful in solidifying the known protein concentrations of A1-B2 (45ul and 65ul of BSA) while providing calculated protein concentrations of two unknown solutions containing egg whites and almond milk (Figure 1, Table 1).

 The BSA standard curve graph presented a high ${\mathrm{R}}^2$

of 0.98799 meaning absorption at 595nm vs. protein quantity (ug) was strongly related increasing the reliability of the graph (Figure 1). Samples A1 and A2 presented similar protein concentrations of 1.426mg/ml and 1.471mg/ml; these two concentrations were fairly close to the known protein concentration of 1.38mg/ml (Figure 1, Table 1). Known samples B1 and B2 also portrayed similar protein concentrations of 1.382mg/ml and 1.371mg/ml; these two protein concentrations were nearly identical to the known protein concentration of 1.38ug/ml (Figure 1, Table 1). Samples B1 and B2 (65ul) portrayed a protein concentration average of 1.38mg/ml which was identical to the given concentration of 1.38mg/ml in comparison to the average of A1 and A2 (1.45mg/ml), which was more varied from the expected concentration (Figure 1, Table 1). The experimental concentrations of A1-B2 were fairly similar to the known concentration of 1.38mg/ml displaying small percent errors of 0% (B1,B2) and 5.07% (A1,A2) increasing the reliability of the BSA standard curve (Figure 1, Table 2).

 The equation of the line (y = 0.0111x + 0.0228) from the BSA standard curve was utilized to determine protein quantities from two unknown samples with concentrations being subsequently calculated (Figure 1, Table 1,2). The almond milk samples (D1 and D2) exhibited protein concentrations of 3.01 mg/ml and 2.88mg/ml with an average of 2.94mg/ml; the protein concentration average of almond milk (2.94mg/ml) was much lower when compared to the higher concentration average of egg whites (C1 and C2) at 36.76mg/ml. The calculated concentration average of egg white was 36.76mg/ml, which was much lower than the expected value of 103.1mg/ml (Table 2, Figure 1). The obtained almond milk concentration (2.94mg/ml) was also lower when compared to the literature value of 4.17mg/ml (Table 2, Figure 1). Both unknown solutions portrayed high percent error values of 29.49% (almond milk) and 67.47% (egg white) expressing high degrees of variance from the expected protein concentration (Table 2, Figure 1).

Figure 1: BSA Standard Curve (1.38mg/ml) Of Varying Volumes At 595nm Demonstrating An ${\mathit{R}}^{\mathit{2}}$

Value Of 0.98799

Table 1: Protein Concentrations (mg/ml) Determined From BSA Standard Curve Of Known And Unknown Solutions

Table 2: Average Protein Concentrations Between Two Known Samples Of BSA Standard And Two Unknown Samples Of Almond Milk And Egg White

Discussion

 The central concept that was emphasized from the results of this experiment was that the Bradford assay and BSA standard curve was accurate in determining the protein concentration of two known samples (BSA of 45ul and 65ul), but inaccurate when quantifying unknown samples of egg white and almond milk (Bio Rad, 1998; Golden Egg, 2018; Silk Products, 2018). The protein concentration of almond milk and egg white appeared to be different from the literature documentation of 4.17mg/ml and 101.3 mg/ml (Golden Egg, 2018; Silk Products, 2018).

 The known sample containing 40ul of BSA standard presented a protein concentration average of 1.45mg/ml, which was fairly similar to the known concentration of 1.38mg/ml as shown by a smaller percent error of 5.07%; the large experimental concentration was most likely caused by an overly concentrated sample (Kruger, 2002). The sample was most likely not diluted exactly to the necessary 5.1ml leading to a concentrated sample misrepresentative of the actual concentration of 1.38mg/ml (Kruger, 2002). The known sample containing 65ul of BSA standard presented a protein concentration of 1.38mg/ml, which was identical to the known concentration of 1.38mg/ml (0% error) increasing the accuracy of the constructed standard curve (Kruger, 2002).

 The experimental egg white sample contained a higher protein concentration average of 36.76mg/ml while almond milk retained a lower concentration average of 2.94mg/ml; this result was expected since Golden Eggs manufacturers have shown that egg whites retained a larger protein concentration of 103.1mg/ml while Silk products contained a lower concentration of 4.17mg/ml (Golden Egg, 2018; Kruger, 2002; Silk Products, 2018). The average concentration of almond milk at 2.94mg/ml was varied from the expected concentration displayed from Silk Almond products at 4.17mg/ml (Silk Products, 2018). A lower experimental protein concentration of 2.94mg/ml most likely indicated that the Bradford assay can be insufficient when binding; the Coomassie dye selectively binds to basic amino acids, which means certain proteins may have not bonded efficiently due to their amino acid sequence lacking basic residues. (Silk Products, 2018; Kruger, 2002). The protein concentration average of liquid egg whites was 36.76mg/ml, which was much lower than the concentration portrayed from the manufacturer, Golden Egg at 103.1mg/ml (Golden Egg, 2018). The lower experimental protein concentration of 36.76mg/ml indicated that the Bradford assay could be ineffective when binding to certain proteins lacking appropriate features such as basic amino acid residues (Golden Egg, 2018; Kruger, 2002).

The unexpected lower protein concentrations expressed in egg whites and almond milk was related to the numerous limitations of the Bradford assay (Golden Egg, 2018, Kruger, 2002, Silk Products, 2018). The Coomassie dye does not bind to free arginine and lysine residues or to peptides smaller than around 3000Da limiting the effectiveness of the assay to quantify all general proteins (Nouroozi et al., 2017; Kruger, 2002). The Bradford assay was also subject to variation in sensitivity between individual proteins; this means that some proteins were more reactive to the dye than others as shown by scientific studies (Bio Rad, 1998; Kruger, 2002). In order to increase sensitivity of the assay, optimum pH conditions for the analyzed sample should be implemented; Variable reactivity with different proteins could have been decreased by adding NaOH to the samples since this has been shown to cause an increase in the proportion of free dye in the blue form leading to further reactivity with proteins as shown by Kruger (2002). While this addition has been shown to improve protein reactivity, optimum pH was strongly dependent on the concentration of the dye and the protein source making it difficult to achieve optimum dye sensitivity unless the protein was thoroughly analyzed (Kruger, 2002). Materials and substances contained in egg whites and almond milk may have interfered with the biochemical process of the Bradford assay causing decreased absorbance values to occur as well (Golden Egg, 2018; Kruger, 2002; Silk Products, 2018). The Bovine serum albumin standard also had an unusually large response to the Coomassie dye leading to an unavoidable underestimation when trying to determine unknown protein concentrations of almond milk and egg whites (Kruger, 2002). This factor was most likely the central error since the reactivity of the initial standard utilized (BSA) was highly varied from the reactivity of other general proteins (Golden Egg, 2018; Kruger, 2002; Silk Products, 2018). Studies have shown that bovine γ-globulin was a more suitable general standard for protein quantification because the dye binding capacity of this protein is closer to the average of most proteins being compared (Kruger, 2002; Stepanchenko et al., 2011).

 This study could be brought into another topical direction by comparing various methods of protein quantification between the Bradford assay, BCA assay, and the Folin-Lowry assay to determine the most effective method for protein quantification of almond milk and egg whites (Stepanchenko et al., 2011). This study could be repeated utilizing the same unknowns of egg whites and almond milk and the acquired experimental protein concentrations could be compared to the literature values of 4.17g/ml and 101.3mg/ml to determine the most effective protein assay method (Stepanchenko et al., 2011).

References

• Golden Eggs, (2018). Retrieved from https://www.nationalegg.ca/
• Bio-Rad Laboratories. (1998) Bio-Rad Protein Assay. LIT33 Rev C. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf

• Chalupa-Krebzdak, S., Long, C. J., & Bohrer, B. M. (2018). Nutrient density and nutritional value of milk and plant-based milk alternatives. International Dairy Journal, 87, 84-92.
• González-González, M., Mayolo-Deloisa, K., Rito-Palomares, M., & Winkler, R. (2011). Colorimetric protein quantification in aqueous two-phase systems. Process Biochemistry, 46(1), 413-417.
• Kruger, N. J. (2002). The Bradford Method for Protein Quantitation. Protein Protocols Handbook, The, 15-22. 
• Nouroozi, R.V., Noroozi, M.V., & Ahmadizadeh, M. (2017). Determination of Protein Concentration Using Bradford Microplate Protein Quantification Assay.
• R., A., S., L., H., & LG. (2018). Unsweetened Vanilla Almondmilk. Retrieved from https://silk.com/products/unsweetened-vanilla-almondmilk
• Stepanchenko, N. S., Novikova, G. V., & Moshkov, I. E. (2011). Protein quantification. Russian Journal of Plant Physiology, 58(4), 737-742. 

Appendix

Sample Calculations

Known Sample A1 Using Standard Curve For Protein Amount:

 0.656 = 0.0111x + 0.0228

0.656 – 0.0228 = 0.6332

0.6332=0.0111x

0.6332/0.0111 = x

x (protein amount ug) = 57.04

Protein Concentration Of Sample A1 Calculation:

Solution diluted to 100ul with water

Known Sample A1 Concentration

Known Sample A = 57.04ug of protein

Amount of Protein Solution = 40ul

57.04ug/40ul = 1.426 ug/ml

Table 3: BSA Standard Curve Measurements Of Varying Volumes At 595nm Diluted To 100ul With DI Water

Average Protein Concentration Calculation

1.426 + 1.471/2 = 1.45mg/ml for 40ul of BSA (A1+A2 Average)

Table 4: Protein Sample Measurements Of 2 Known Solutions (A1-B2) And Two Unknown Samples Of Egg White And Almond Milk (C1-D2)

Concentration Of Egg White Calculation Golden Egg:

Density of egg: 1.031g

50g serving x 1ml/1.031g = 48.496605ml

5000mg/48.496605ml = 103.1g

Concentration Of Almond Milk Silk Products:

1000mg of egg white in 240ml

10000mg/240ml = 4.17mg/ml

Percent Error Calculation:

$\frac{1.45–1.38}{1.38}$

x 100 = 5.07% for 45ul of Bradford known solution (A1 + A2 average)