Spectrophotometry and Protein Quantitation via Bradford Method PDF

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Spectrophotometry and Protein Quantitation via Bradford Method I. Abstract The purpose of this experiment is to estimate the protein concentrations of two unknown stock solutions via the Bradford Method. The Bradford Method is a technique for protein quantitation exploiting the high molar absorptivity of the Coomassie Brilliant Blue G-250 dye-protein complex. The first phase of the experiment involves calibrating two different pipettes using the known density of dH 2O (1g/mL). The second phase involves generating a standard curve using a known concentration of BSA (1µg/µL) to estimate unknown protein concentrations. The final phase includes making dilution series for both unknown stock solutions and estimating protein concentrations based on absorbance values. Both unknown 1 and unknown 2 stock solutions were found to be highly concentrated with concentration values of 6.79µg/µL and 14.46µg/µL, respectively. II. Introduction Inside the biochemistry laboratory, chemical compounds that are vital to allowing organisms to live are carefully observed and studied. One such molecule necessary for organisms to live are proteins. As many molecules in the laboratory are too small for the naked eye to detect, careful detection methods are implemented. The Spec-20 spectrophotometer is an instrument in the laboratory that measures the absorbance of various solutions ranging from wavelengths (λ) at 340nm to 950nm. Spectrophotometers directly measure the intensity of light transmitted through a sample. For this specific experiment, a wavelength of 595nm will be chosen. It is vital to calibrate, or “blank”, the spectrophotometer with a cuvette containing the solution used with the molecule being studied, however, in absence of the analyte where the absorbance value will read 0.00 (0% light absorbed by the sample). It is also necessary to calibrate the machine by leaving the cuvette slot empty (or using an opaque cuvette) and setting the machine to 100% absorbance. Proteins contain a unique arrangement of amino acids covalently linked in a linear structure through peptide bonds. Proteins make up approximately 10-50% of the dry weight of most cells and perform a vast amount of actions required for the cell to live and reproduce. Unfortunately, samples containing a specific protein of interest also include a large amount of other cellular components. To avoid this problem, biochemists record the total protein content in a sample and express results as “mg protein/ml solution.” In biochemistry, it is imperative to be able to accurately estimate concentrations of proteins, as they are vital for numerous cell functions. In order to do this, methods (such as the Bradford Method) exploit the interaction between proteins and compounds that undergo color change when protein is present. The Bradford Method is an assay commonly used in biochemistry to measure concentrations of protein in solution. This method is dependent on the interaction between the dye, Coomassie Brilliant Blue G-250, and protein which produced a blue color ranging in depth when bound to protein. The dye is red when free in solution and undergoes a color change to blue when bound to protein. The protein-dye complex has a high molar absorptivity at wavelength 595nm. The Bradford Method is commonly used due to its simplicity and its highly sensitive nature. III. Materials and Methods In order to ensure the accuracy and precision of future measurements, two air-displacement pipettes were examined and calibrated. A P-200 (range: 20-200µL) and a P-1000 (range: 100-1000µL) were chosen to be calibrated. The pipettes were obtained and set to their lowest volume, taking care not to set the pipette above or below their indicated range. A small beaker was obtained and filled with dH 2O. A weigh boat was placed on an analytical balance and zeroed. The P-200, set at its lowest volume within its range, was used to pipet the set volume of liquid into the weigh boat and the weight was recorded. This was done three times while the balance was zeroed between each measurement. The P-200 was then set to

its highest volume within its range and the set volume of liquid was pipetted into the weigh boat and the weight was recorded. This was done three times while the balance was zeroed between each measurement. The same procedure was repeated for the P-1000. While the calibration of the pipettes was being done, the Spec-20 spectrophotometer was turned on to warm up the bulb. This was done 30 minutes prior to using the machine to ensure it would work properly. To accurately estimate the concentration of unknown protein samples, a concentration standard was used. In this case, a 1mg/mL concentration of BSA (bovine serum albumin) was used to generate a standard curve. Samples used for a BSA standard curve ranged from 0-100µg of protein using 1.0mg/mL (1.0µg/µL) BSA. The amount of BSA for samples used to generate the standard curve was: 0, 10, 20, 40, 60, 80, and 100µg. Standards were prepared by adding the appropriate amount of stock protein solution to a reaction tube and adding enough ddH2O to bring the final volume to 100µL. Duplicates of each BSA sample amounts were set up. 5.0mL of Bradford reagent was then added to each reaction tube containing varying amounts of BSA, immediately vortexed, and left to sit for 5 minutes for the reaction to complete. The Spec-20 spectrophotometer was then set to wavelength 595nm and blanked with the sample containing no protein. The absorbance values of each reaction tube was recorded. Excel was then used to construct a standard curve to determine the concentrations of the unknown samples. The amount of sample from stock solutions Unknown 1 and Unknown 2 and amount of ddH 2O were determined to prepare the following dilutions: 1/2, 1/10, and 1/50. The dilutions were then prepared based on previous calculations. The undiluted samples and the dilutions were kept on ice. Samples for unknowns were prepared by adding 100µL of each undiluted sample and 100µL of each dilution series to a reaction tube. Duplicates for each unknown sample were set up. 5.0mL of Bradford reagent was added to each reaction tube, immediately vortexed, and left to sit for 5 minutes for the reaction to complete. Absorbance values were then recorded for all undiluted samples and dilutions. After completion, all reagents, used and unused, were discarded in the large disposal bin and the work area was thoroughly cleaned. IV. Results Figure 1.1 Pipettes Weight #1 P-200 Lower P-200 Upper P-1000 Lower P-1000 Upper

Weight #2

Weight #3 0.0388g

Average Weight 0.0395g

0.04g

0.0396g

0.1980g

Variance 2.5 x 10-7g

Standard Deviation 5 x 10-4g

0.1975g

0.1928g

0.1961g

1.6 x 10-5g

4.1 x 10-3g

0.1048g

0.1045g

0.1038g

0.1044g

5.3 x 10-7g

7.3 x 10-4g

1.0001g

1.0030g

1.0007g

1.0013g

4.7 x 10-6g

2.2 x 10-3g

Figure 1.1. Depicted above are the weights of three trials measuring dH2O pipetted for the upper and lower bounds of the P-200 (20-200µL) and the P-1000 (100-1000µL). Using the density of dH2O (1g/mL), the accuracy of each pipette could be determined. Figure 1.2 Amount of BSA (µg) 0 10 20 40 60

A595 #1 0 0.07 0.15 0.225 0.31

A595 #2 0 0.07 0.13 0.235 0.31

Average 0 0.07 0.14 0.23 0.31

80 100

0.39 0.42

0.35 0.44

0.37 0.43

Figure 1.2. Depicted above are the absorbance values of the differing amounts of BSA used to generate a standard curve. The absorbance values were taken in duplicates to get a more accurate interpretation of the standard curve used to approximate unknown protein concentrations.

Graph 1.1

Absorbance (595nm)

BSA Standard Curve 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

f(x) = 0 x 0 R² = 0.09 10

20

30

40

50

60

70

80

90

100

Amount of BSA (µg)

Graph 1.1. Depicted above is the standard curve generated from taking the average of two absorbance values from differing amounts of BSA. The standard curve was made taking the first three values so that they fell within the linear range of the Spec-20. The absorbance values corresponding to differing amounts of BSA can be observed in Figure 1.2. Figure 1.3 Dilution Factor Unknown 1: Stock Unknown 1: 1/2 Unknown 1: 1/10 Unknown 1: 1/50

A595 #1

A595 #2

1.1

0.95

0.93

0.93

0.6

0.62

0.09

0.1

Dilution Factor Unknown 2: Stock Unknown 2: 1/2 Unknown 2: 1/10 Unknown 2: 1/50

A595 #3

A595 #2

1.1

1.1

1.0

1.1

0.7

0.68

0.205

0.2

Figure 1.3. Depicted above are the absorbance values recorded for each dilution factor of unknown solutions 1 and 2. Dilutions were made according to calculations using the sample dilution calculation below. Sample Dilution Calculation - (Final Rxn Volume) x (1/DF) = Volume of protein solution needed o DF = Dilution factor (undiluted = 1, 1/50 dilution = 50)

Sample Protein Concentration Calculation -

( µg of protein used ∈sample) x DF ( µL of sample used ∈assay) o o

DF = Dilution factor (undiluted = 1, 1/50 dilution = 50) µL of sample used = 100µL

V.

Discussion While performing the calibrations of the pipettes, it was found that the instruments were most accurate towards their upper volume limit within their range. The P-200 was found to be fairly inaccurate towards its lower bound (20µL). Theoretical value of the lower bound of the P-200 should be 0.02g. This error could be a result of possible tampering of the volume adjustment knob. The P1000 was found to be fairly accurate at both its lower (100µL) and upper (1000µL) bounds. Both stock solutions containing unknown concentrations of protein were found to be highly concentrated. Utilizing the equation derived from the BSA standard curve in Graph 1.1 and the sample protein concentration equation, it was found that the concentration of unknown solution 1 was approximately 6.79 µg/µL and the concentration of unknown solution 2 was approximately 14.46 µg/µL. These concentration values were approximated using the A 595 values of the unknown solutions that fell within or came close to falling within the linear range of the BSA standard curve. However, the A595 value for unknown 2 did not fall within the true linear range of the standard curve, therefore the concentration value is a rough estimate. The high concentrations of each unknown stock solution is logical due to the fact that their A 595 values were closest to the linear range of the BSA standard curve at the 1/50 dilution.

VI. Questions 1) The average of the measurements of the lower bound of the P-200 were 0.0195g off of the theoretical weight of 0.02g, rendering it almost a full 20µL off. The average of the measurements of the upper bound was 0.0039g off of the theoretical weight of 0.2g, making it fairly accurate towards its upper bound. The average measurements of the lower bound of the P-1000 were 0.0044g off the theoretical weight of 0.1g, while the average measurements of the upper bound was 0.0013g off the theoretical weight of 1.0g, rendering the P-1000 fairly accurate for its full volume range. 2) The P-200 was fairly precise at both its lower and upper volumes within its range, however, was only accurate at its upper bound. The P-1000 was both accurate and precise at both its upper and lower bounds. More measurements would be helpful in determining the accuracy and precision of each pipette. 3) All of the samples made for the standards should not be used to construct the standard curve. Only the first few samples fall within a true linear range, while every other sample deviates from a true linear curve. Using every sample would result in an inaccurate standard curve, rendering unknown protein concentrations estimations inexact. 4) Serial dilutions need to be prepared for unknown samples due to the uncertainty of its original concentration. A dilution must be achieved to yield an absorbance value that falls within the true linear range of the BSA standard curve. 5) The blank solution consists of 5.0mL of Bradford reagent and 100µL dH2O. It is important to have a blank solution in order to calibrate the spectrophotometer with a solution containing no protein so that an accurate absorbance value can be achieved when protein is present in solution. 6) The Bradford assay obeys the Beer-Lambert Law over concentrations 0-20µg. 7) This is a good reference protein due to the Bradford reagent’s high sensitivity, however, the protein includes many of the amino acids lysine and arginine which slightly affect dye binding....