Practical 3 Bradford assay PDF

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Practical 3: Determining the Specific Activity of Yeast Invertase – the Bradford AssayIntroduction For measuring the specific activity of the invertase enzyme the amount of protein is important because you are not working with a pure invertase preparation; therefore not all of the protein present in...

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Practical 3: Determining the Specific Activity of Yeast Invertase – the Bradford Assay Introduction For measuring the specific activity of the invertase enzyme the amount of protein is important because you are not working with a pure invertase preparation; therefore not all of the protein present in the crude mixture is the enzyme of interest. Remembering from the last practical: “The concept of activity of an enzyme is extended further when we consider the actual amount of protein in the enzyme mixture. Remember that (generally) all enzymes are proteins but not all protein in a mixture is the enzyme of interest (there may be other enzymes and non-enzyme proteins in a crude protein extract). Thus, the term specific activity refers to the activity of the enzyme of interest per amount of total protein in the mixture. This reference to the amount of total protein provides a convenient way of defining and comparing the purity of enzyme preparations. It allows comparisons between enzyme preparations produced from different batches or different experiments. Specific activity is usually expressed as units per mg of protein (i.e. µmol min-1 mg-1 or U mg-1).” There are numerous methods used to determine the concentrations of proteins in a solution, including the A280 Assay (which you learnt in your introduction practical) and the Lowry Assay which you will use in later experiments. Often though, an accurate determination of protein concentration needs to be done in the presence of solubilising detergents. This is because many proteins can’t be dissolved easily in conventional aqueous solutions. Detergents are also necessary in some circumstances to ensure a protein maintains its shape, as many proteins will denature without the presence of surfactants to shield a protein’s hydrophobic regions. This is particularly true for cell membrane-bound proteins. But the presence of detergent can also be detrimental as the function of many proteins can also be diminished in their presence. In this experiment, you will be provided with a sample of the yeast invertase protein you used in the previous practical. A standard graph will be prepared using albumin (bovine serum albumin, BSA) as a known standard or reference protein. The protein content of 1 in 5 and 1 in 10 dilutions of your unknown yeast invertase sample will be assayed and, along with the enzyme activity results of the previous experiment, you can then determine the specific activity of the enzyme. In this practical, you will be using the Bradford protein assay. The Bradford procedure was first described by Marion Bradford in 1976 and is now, along with the Lowry Assay, one of the most widely used protein assays (Bradford M., Anal. Biochem 72.1-2 (1976): 248-254.]. The Bradford assay makes use of a dye called Coomassie Brilliant Blue G -250. Under acidic conditions, this dye has a red-brown colour; however, in the presence of relatively low amounts of protein, the dye shifts to blue in its colour. This colour change can be readily measured by taking absorbance readings at 595nm (i.e. an A595 reading). Whether or not yeast invertase protein is better solubilised in the presence of detergent will also be assessed. When obtaining enzyme samples, such as yeast invertase, the protein itself needs to be made in culture, in this case a yeast culture. Large quantities of yeast cells are typically grown in bioreactors. The yeast cells are then lysed and proteins are released. Some purification steps can then be used to isolate particular enzymes or proteins. This purification step is often less than ideal, particularly if large concentrations of the enzyme are desired, as there are often many impurities leftover in the final product, including cell

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membrane fragments. These cell membrane fragments can have proteins bound to them and if enzymes are embedded in membranes, they can be shielded from dyes, such as the Coomassie dye used in the Bradford assay. Here, we will determine whether a detergent can solubilise more of the protein than can be measured from a cell lysate product with all its leftover membrane fragments. The hypothesis being tested is: That more protein will be measured in aqueous samples in the presence of detergent than without. As detergents are surfactants, just like cell membrane phospholipids, it could be that the presence of the detergent itself will act to shield the protein. As detergents are known to influence the responses of Bradford assays, you will need to perform separate standard curves for samples with and without detergents. To do this, standard curves using a 0.01% concentrations of the detergent Triton-X 100 will be compared to using pure water. This practical will also introduce you to using micro-plate readers for measuring spectral absorbance changes. Micro-plate readers are valuable tools as they allow for multiple simultaneous measures that can then be compared and correlated. They also use less reagents that standard 3 mL cuvette measures. A typical micro-plate has 96 wells, its rows are labelled with letters A-E and its columns are numbered 1-12. (see Figure below).

1 A B C D E F G H

2

3

4

5

6

7

8

9

10

11

12

Sample Only

2

Preparation of protein standards for standard curve of protein content 1. Obtain a 96 well plate. 2. In wells A1-A8 and B1-B8 set up dilutions to contain 0, 1.25, 2.5, 5, 10, 15, 20, and 25 µg/mL using a stock BSA stock solution of 2 mg/mL. The 0 µg/ml protein wells (A1 and B1) will be the blanks (zero absorbance) for spectrophotometric analysis. To these wells simply add 150 µL of dH2O. You will need to dilute the stock BSA sample in order to be able to create the correct standards that are in the µg/ml range. To do this, follow these steps: a) Label 7 Eppendorf Tubes S1,S2,…S8. b) Prepare dilutions according to the table below and calculate the final concentration of each of standard.

Tube Label

Vol of H2O (µL)

Vol of 2mg/mL BSA solution (µL)

Vol of other dilution to add?

S1

790

10

0

25

S2

990

10

0

20

S3

1588

12

0

15

S4

500

0

500µL of tube S2

10

S5

500

0

500µL of tube S4

5

S6

500

0

500µL of tube S5

2.5

S7

500

0

500µL of tube S6

1.25

S8

500

0

0

Final Conc (µg/mL)

0

c) Mix the contents of each tube by inversion. Add 150 µL of each of the above standard solutions to the appropriate wells, A1-A8, and duplicate row B1-B8, of the micro-plate according to the template below.

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Dilution of the unknown protein samples for analysis As different assays have different sensitivities or different ranges of measurement, dilution of the samples is often required to capture the optimal range for a particular assay (see below). The unknown protein samples will be assayed at dilutions of 1 in 5 and 1 in 10.

sampleB →

sampleA →

Absorb ance (nm)

Amount (mmol)

The standard curve is only linear over a certain range. When there are larger amounts of standard a linear relationship no longer exists. So, if the absorbance reading of a sample is greater than the absorbance reading of the highest standard then you cannot read the amount from the graph. In the diagram to the left, an amount for sample A can be read from the graph but an amount for sample B cannot.

3. Set up 2 eppendorf tubes and label one of them “1 in 5” and the other “1 in 10”. Pipette 800 µL H2O into the “1 in 5” dilution tube and 900 µL into the “1 in 10” dilution tube. 4. Add 200 µL of your unknown yeast invertase sample to the “1 in 5” dilution tube and 100 µL into the “1 in 10” dilution tube. 5. Mix by inversion. Then add 150 µL of each to the appropriate wells in Row C of the micro-plate (see Template below).

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Preparation of detergent standard samples. 6.

a) Label 7 Eppendorf Tubes ST1,ST2,…ST8. b) Prepare the following dilutions. Note that these tubes are labelled “ST” in order to denote the presence of Triton-x 100.

Tube Label

Vol of H2O (µl)

Vol of 2mg/mL BSA solution (µl)

Vol of 0.1% triton-X 100

Vol of other dilution to add?

ST1

710

10

80

0

25

ST2

890

10

100

0

20

ST3

1428

12

160

0

15

ST4

450

0

50

500 µL of tube ST2

10

ST5

450

0

50

ST6

450

0

50

ST7

450

0

50

ST8

450

0

50

Final Conc (µg/mL)

500 µL of tube ST4 500 µL of tube ST5 500 µL of tube ST6 0

5 2.5 1.25 0

c) Mix the contents of the standard tubes gentle inversion – avoid frothing. Add 150 µL of each standard solution to rows E and F in your micro-plate, according to the template below – again avoid introducing bubbles to the wells (bubble = bad result!!). 7. Set up 2 eppendorf tubes and label one of them “UT1 in 5” and the other “UT1 in 10”. Pipette 700 µL H 2O into the “1 in 5” dilution tube and 800 µL into the “1 in 10” dilution tube. Then add 100 µL of 0.1% Triton-X 100 to each tube. 8. Add 200 µL of your unknown yeast invertase sample to the “1 in 5” dilution tube and 100 µL into the “1 in 10” dilution tube. 9. Vortex both tubes gently to mix the solution. Then add 150 µL of each to the appropriate wells in Row G of the micro-plate (see Template below).

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Template: Your 96 Well plate should have the following samples in each well: 1 A

B

C D E

F

G

2

S2 S1 20.00 25.00 µg/ml µg/ml S1 S2 2 µ U 1 Standard (no

3

4

S3 15.00 µg/ml S3

S4 10.00 µg/ml S4

Triton-X 100)

S 2 µ S 2 µ U 1

5

6 S6 2.50 µg/ml S6

S5 5.00 µg/ml S5

Vol of sample in well (mL)

7

8

9

1 0

concentration µg/mL µg protein

25

S2

0.15

20

S3

0.15

15

2.25

0.7341

S4

0.15

10

1.5

0.6286

S5

0.15

5

0.75

0.4461

S6

0.15

2.5

0.375

0.4084

S7

0.15

1.25

0.1875

0.3806

S8 (Blank)

0.15

0

0

0.3018

U = Unknown

3.75

(Average) A595

0.15

S = Standard

1 2

S8 0.00 µg/ml S8

S7 1.25 µg/ml S7

S1

H

11

3

0.8788 0.7936

T = Triton-X 100

Add Bradford Reagent 10. Add 150 µL of the Coomassie dye solution to each well. Gently aspirate with your pipette to ensure mixing each time. Measure using microplate reader 11. Set the micro-plate spectrophotometer to absorbance mode to be read at 595 nm. Get an electronic copy of the data and upload it to your bench-top computer/tablet. 12. Using Excel, plot your standard curves. Do a regression analysis on the data (see below).

Complete this Table for your samples without detergent.

Complete this Table for your samples that contain detergent

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Standard (+Triton-X 100)

Vol of sample in well (mL)

concentration µg/mL µg protein

(Average) A595

ST1

0.15

25

3.75

0.8721

ST2

0.15

20

3

0.8127

ST3

0.15

15

2.25

0.7767

ST4

0.15

10

1.5

0.6574

ST5

0.15

5

0.75

0.5152

ST6

0.15

2.5

0.375

0.4573

ST7

0.15

1.25

0.1875

0.4092

ST8 (Blank)

0.15

0

0

0.3287

Plotting a Standard Curve Using Excel Spreadsheet: Using the standards data construct the standard curve with Excel and report the equation of the line of best fit (the “regression line”). [Note: this exercise introduces the use of Excel in data analysis.] Introductory videos are available on UTS Online. Your demonstrators can help you find them. Please watch these videos. They are a great introduction to Regression Line analysis. Results of Data Analysis: Regression line (line of best fit) equation for you non-detergent standard: y =0.3469 +0.1521 x

An equation can lose its meaning if it’s not put in context. Please fill in the following blanks to define the equation and its variables. The dependent variable on the y-axis are the ___Absorbance___ values, whereas the independent variable on the x-axis is the ___Mass____ of protein (BSA). Assessing the Goodness of Fit: The next step in analysing the data in Table 1A is to determine whether those results are reliable and reflect the linear relationship described in Beer’s law. In this session we will examine the R2 or the “coefficient of determination” of the line of best fit. The R2 takes on a value between 0 and 1 where a value of 1 means a perfect fit and all of the variations in absorbance can be explained by changes in protein content. A value of 0 would mean that 7

changing the protein content doesn’t appear to affect absorbance at all. Therefore, in defined conditions, a high R2 (>0.95) should be observed for the line of best fit. R2 for the line of best fit drawn above: ¿ R2=¿ 0.951¿ Comment on the reliability of the data recorded in your non-detergent Standard curve. The R^2 of the nondetergent standard curve is >0.95 therefore, can be considered reliable List 3 factors that could reduce the reliability of the standard curve. -i n t e r f e r i n ga g e nt s -Sa mp l ep r e p a r a t i o n -Pr o t e i n t o pr o t e i nv a r i a t i o n s( n op r o t e i n d y ec o mp l e xf o r me d )

Save your Excel File with your 2 Standard Curves with your Name and Student ID in the Title.

Unknown protein sample – no detergent Calculation Steps

Units

Unknown Yeast Invertase Sample Dilution 1 in 5

1 in 10

(Wells C1-3)

(Wells C6-8)

a.

A595

-

U1:5 (C1)

U1:10 (C6)

b.

A595

-

U1:5 (C2)

U1:10 (C7)

c.

A595

U1:5 (C3)

U1:10 (C8)

d.

Average A595

e.

Amount of protein

-

0.5679

0.4282

g

1.4530

0.5345

mL

0.15

0.15

[use standard curve] f.

Volume of unknown sample dilution tested

8

g.

Concentration of diluted protein (e/f)

g/mL

9.687

3.563

h.

Concentration of original (undiluted) protein sample.

g/mL

48.35

35.633

Adjust for Dilution factors (g. times dilution factor)

9

Unknown protein sample in Triton-X 100

Calculation Steps

Units

Unknown Yeast Invertase Sample Dilution 1 in 5 (Wells G1-3)

1 in 10 (Wells G6-8)

a.

A595

-

UT1:5 (G1)

UT1:10 (G6)

b.

A595

-

UT1:5 (G2)

UT1:10 (G7)

c.

A595

UT1:5 (G3)

UT1:10 (G8)

d.

Average A595

e.

-

0.5729

0.4801

Amount of protein [use standard curve]

g

1.261

0.609

f.

Volume of unknown sample dilution tested

mL

0.15

0.15

g.

Concentration of diluted protein (e/f)

g/mL

8.447

4.06

h.

Concentration of original (undiluted) protein sample. Adjust for Dilution factors (g. times dilution factor)

g/mL

42.235

40.6

Interpretation of results 10

Q1. What was the calculated protein concentration (in g/mL) of your unknown sample with and without Triton-X 100 (from the 1:10 dilution samples)?

No Detergent:_ With Triton-X 100_

36.633 40.6

Q2. Calculate the concentration of the unknown yeast invertase samples in mg/ml.

Calculated Protein Concentration (No Detergent) =

0.0366

mg/mL.

Calculation Protein Contentration (in presence of Triton-X 100) = mg/mL

0.0406

Q3. Comment on the reliability of the unknown sample estimations both with and without detergents? Can you make a determination as to which is more accurately reflects the true amount of protein? Since both graphs produced a R^2 value >0.95 indicating that both these graphs are accurate. The function of the detergent should have lysed and released the proteins from the yeast invertase and so, should show a higher value to the protein concentration of the two values (with detergent)

Using the protein concentration you trust more (considering your answer to Q4 above) complete the following calculation:

Units of Invertase activity/mL (in U/mL)

49U/mL

Protein concentration (in mg/mL)

0.0406mg/mL

(From Practical 2)

Specific Activity (in U/mg of protein) 1206.90 U/mg (U/mL  mg/mL will cause the ‘mL’ unit to be cancelled out leaving units of U/mg U/mL  mg/mL = U/mL x mL/mg ) 11

When you have completed your calculations, standard curves and answered your questions: 

Save this Document and your Excel file and upload a copy to your demonstrators via this form by Friday 5pm

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