Lab 2 Report PDF

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Lab 2 Report...

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LAB 2 OUTLINE: PROTEIN QUANTIFICATION: LOWRY AND BRADFORD ASSAYS Objective: The objective of this lab is to determine the total protein concentration of our E. Coli BL21 (DE3) lysate using two colorimetric techniques: the DC Protein Assay and the Bradford Assay.

Principle of Methods:

Lowry (DC) Protein Assay: This is a two-step process that has a change in color once it reaches 750 nm. Alkaline copper solution and 5% Reagent AS will be added to the protein sample. The copper found in the solution will form a complex with the amine group of the protein’s amino acids if it finds itself in an alkaline environment. Binds equally with all amino acids, leading to less protein-to-protein vulnerability. After this, Folin Reagent is added, the copper treated protein reduces this reagent. A reduction of the Folin Reagent produces a blue color. After the addition of Reagent B, color production will continue for up to two hours. (Lab Manual).

Standard Curve: The standard curve is the correlation between the absorbance of samples prepared and the unknown samples of the E. coli.

Bradford Assay: Coomassie Brilliant Blue binds to basic and aromatic amino acids. Does not bind all proteins equally, leading to high protein to protein variability. As protein concentration increases, the color shifts from red to blue. A shift in absorption from 465 nm (unbound dye) to 595 nm (bound dye) allows the amount of protein to be determined by spectrophotometer. (Lab Manual).

Materials: 

E. coli BL21 (DE3) lysate pET-6XHis-GST-EGFP.



Alkaline Copper solution



5% SDS (Reagent AS)



Folin Reagent (Reagent B)



Coomassie Blue Brilliant



BSA

Results: Table 2-4 BSA (mg/ml) 0 0.3 0.3 0.6 0.6 0.9 0.9 1.2 1.2 1.5 1.5 1.8 1.8

Sample # Blank 1 2 3 4 5 6 7 8 9 10 11 12

Absorbance (750 nm) 0 0.094 0.099 0.173 0.175 0.242 0.104 0.299 0.318 0.395 0.409 0.272 0.441

Table 2-5 Absorbance

Amount

Dilution

Final

Average protein

(750 nm)

(mg/ml)

Factor

Concentration

Concentration (mg/ml)

13

0.031

0.015

25

0.375

14

0.017

-0.051

25

-1.275

Sample #

1.37

15

0.101

0.35

5

1.75

16

0.103

0.36

5

1.8

17

0.184

0.74

2.5

1.85

18

0.181

0.73

2.5

1.825

19

0.275

1.17

1.67

1.95

20

0.253

1.07

1.67

1.79

21

0.322

1.39

1.25

1.73

22

0.342

1.49

1.25

1.86

Table 2-6 Bradford

Lysate

Absorbance

Dilution

Reagent

(uL)

(595 nm)

Factor

Blank

1 ml

0

0

-

-

1

1 ml

1

0.049

20

0.98

2

1 ml

2

0.172

10

1.72

3

1 ml

4

0.322

5

1.61

4

1 ml

5

0.376

4

1.5

5

1 ml

10

0.662

2

1.3

Cuvette #

Value 2-1: 3.5 ml Value 2-2: 1.37 mg/ml Value 2-3: 1.4 mg/ml Value 2-4: 1.396 mg/ml Equation 2-1: y= 0.2111x + 0.0278

Final

Average

Concentration Concentration -

1.4

BSA Standard Curve 0.5 0.45

Absorbance (750 nm)

0.4

f(x) = 0.21 x + 0.03 R² = 0.81

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

BSA Amount (mg/ml)

Discussion: The purpose of this laboratory experiment was to determine the total protein concentration using two colorimetric techniques: the DC Protein Assay and the Bradford Assay. After looking at the data results, we can clearly see that both techniques provide us quite very similar results when measuring the absorbance and the BSA amount. Taking a closer look at the DC Assay in Table 2-5, I did notice, in my opinion, an erroneous calculation. The Amount value of -0.051, it is quite shocking to obtain a negative value in these sorts of experiments when measuring physical data, although I have not attended the online lab for this experiment due to having another class during that period I can understand my confusion on this part....