4PY013 MBL Protein Assay Protocol 2016 PDF

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4PY013 Molecular Basis of Life PRACTICAL PROTEIN ASSAY

Contents

Page

Foreword

2

Safety information

4

Biuret method

5

Folin method

7

Calculations

8

Statistical analysis

10

Assessment questions

11

MCQ answer grid

13

1

FOREWORD A. WHAT YOU WILL NEED: 1. LABORATORY COAT (no entry to any practical without one of these). 2. HARD BACKED LABORATORY NOTEBOOK This is to be used to record all raw data and will serve for all practical classes. 3. BLACK PERMANENT MARKER PEN B. WHAT YOU MUST DO: 1. Perform the practical INDIVIDUALLY, do NOT work in pairs. 2. Experimental write-up. All the particulars 1. - 11. should be covered. 1 Name 2. Date 3. Title of Experiment 4. Abstract - This should be no more than fifty words. The abstract should contain a summary of the purpose of the experiment, the methods used, the results obtained, and any conclusions drawn. Usually two to three sentences should be sufficient. 5. Introduction - Outline very briefly the objectives of the exercise and the theory behind the experiment. Include chemical structures and reactions where appropriate. 6. Materials and Methods - Do not copy out the experimental sheet you have been given. Cross refer to this and report any changes which may have been made in the experimental procedure or conditions. 7. Results - Where possible all results must be tabulated. All tables must have a title, and units (if any) must be included in the table column or row headings. If you use 'blanks', include these in any protocols you produce. 8. Calculations - Where calculations are included, only one sample for each type of calculation is required. For the remaining calculation record only the results of the calculation in a table. 9. Graphs - Must always be HAND – DRAWN and have a title. Axes must be labelled and appropriate units included. The scale used must be sensible i.e., it can easily be translated. Do not choose scales simply on the basis that the graph fills the paper. Depending on the results, draw the best fit straight line or curve. The zero (0,0) point should have the same weighting as other points when drawing best fit lines on graphs.

2

10. Discussion - Discuss the result and pattern of results you achieve. Try and relate these to accepted theory or literature results, indicating where there are differences (if any). Try to highlight any weaknesses, sources of error etc., in your observations and offer explanations. 11. References - Cite any sources of information.

3. Handing in work (and the late marking penalty). You must hand in your work either at the end of the class when directed, or by the date given to you by your demonstrator. The hand in dates for your group will be posted on WOLF. LATE WORK will be assessed, but will be subject to a minimum pass grade. Hand in your: 1. Tables of results (absorbance values and calculated protein amounts) 2. Graphs 3. Values for X and Y by the two methods 4. SEM calculations 5. MCQ answer grid

C. WHAT YOU NEED TO KNOW: Assessment scheme 1. The practical is MANDATORY and contributes towards the 40% overall grade. It is also a COMPONENT, which means you must pass this in order to PASS the module OVERALL. 2. The grade for the practical will be based on the 10 MCQ assessment questions. 3. Absence from EITHER exercise FOR WHATEVER REASON will necessitate retaking the missing practical(s) WHEN NEXT AVAILABLE (Semester 1, of the next academic year). 4. Assessment is by continuous assessment. There is no resit examination facility for practical work.

3

Safety Information for Protein Assay Chemical/ Material

Hazard

Precautions/ Contamination

Disposal

Biuret reagent

Caustic

Wash down drain with plenty of tapwater

Alkaline copper solution

Caustic

Wash off skin with copious quantities of water; if splashed in eyes irrigate thoroughly and seek medical attention if irritation persists. Wash off skin with copious quantities of water; if splashed in eyes irrigate thoroughly and seek medical attention if irritation persists.

Bovine serum albumin solutions (various concentrations)

May cause allergic reactions in sensitive individuals

Sensitive individuals are advised to wear protective gloves

"

Folin-Ciocalteu reagent (contains hydrochloric and phosphoric acids)

Corrosive

Safety specs. and disposable gloves are recommended. If splashed on skin wash off with copious quantities of water. Medical attention may be required if splashed in eyes.

"

"

Risk assessment - Other than the hazards described in the COSHH information given above there are no hazardous procedures involved in this practical .

4

PROTEIN ASSAY BY BIURET AND FOLIN-CIOCALTEAU METHODS Introduction Quantitative measurement (assay) of protein is one of the most common techniques employed by bioscientists. For example, protein determination is essential for procedures such as enzyme purification or the isolation of sub-cellular fractions and organelles. The protein content of agricultural products is routinely measured, whilst the protein concentration of body fluids is often monitored as an indicator of certain clinical conditions. Thus, virtually all research, industrial and hospital laboratories carry out protein assays at some time. We are going to measure protein by two methods. First, the Biuret method and next week the Folin assay. The two vary in sensitivity and thus in application. The Biuret assay utilises conditions of low [H+] and CuSO4 to produce a co-ordinate complex between the metal ion and consecutive nitrogen atoms in the polypeptide backbone, which results in a deep violet colour (see Fig.1). The Folin reagent interacts with sidechains of tyrosine residues in the protein, again in an alkaline copper environment, resulting in a coloured product, which can be measured spectrophotometrically. Fig.1. N

H2N

NH2 O

O

Cu2

O

O H2N

N

NH2

Materials and safety information Please refer to sheet on page 4.

5

Experimental The Biuret Method Set up tubes and make additions according to the following protocol. Test Tube Number ADDITION (cm3)

1,2

3,4

5,6

7,8

9,10

11,12

13,14

16,17

15

18

19

Water

-

0.1

0.2

0.3

0.4

0.5

0.2

0.2

0.6

Unknown X

-

-

-

-

-

-

0.4

-

-

0.6

0.5

0.4

0.3

0.2

0.1

-

0.4 -

-

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

Unknown Y Standard Protein (5 gdm-3) Biuret Reagent (dark blue) Absorbance 550nm

0

Average

Mix, allow to stand for 30 min. Then read absorbance against the blank (i.e. set zero on the spectrophotomer with the contents of tube 19) at 550 nm. Report 1. Draw up tables of results (do not copy the above protocols) showing the concentration of protein and their respective absorbance values. 2. Draw a calibration curve graph for the Biuret protein assay. HAND DRAWN GRAPHS ONLY PLEASE 3. Determine the concentration of the unknown proteins X and Y. 4. Estimate the Standard Error of the Mean of the triplicate values for X and Y. 5. The colour reaction is in part a chelation. Look up and draw the structure of the chelating agent EDTA, which can be used as an anticoagulant and show how it interacts with Fe2+.

6

The Folin Method Note X/20 means sample X from last week diluted 20 times. Ditto Y/20.

Test Tube Number ADDITION (cm3)

1,2

3,4

5,6

7,8

9,10

11,12

13,14

16,17

15

18

19

Water

-

0.1

0.2

0.3

0.4

0.5

0.2

0.2

0.6

Unknown X/20 Unknown Y/20 Standard Protein (0.2 gdm-3) Alkaline Copper (light blue)

0.6

0.5

0.4

0.3

0.2

0.1

0.4 -

0.4 -

-

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

Mix well, warm for 30 min. at 400C then remove tubes and add … Folin Reagent

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

Absorbance 660nm

0

Average Mix, allow to stand on the bench for at least 15 min. Then read absorbance against the blank (tube 19) at 660 nm.

Report 1. Draw up tables of results (do not copy the above protocols) giving the concentration of protein and their absorbance values. 2. Draw by hand a calibration curve graph for the Folin protein assay. 3. Determine the concentration of the unknown proteins X/20 and Y/20. 4. Estimate the Standard Error of the Mean of the triplicate values for X/20 and Y/20. 5. Examine your results for both Biuret and Folin and use them to answer the 10 multiple choice assessment questions.

7

Protein Assay calculations: Biuret: Plot Absorbance 550nm on y-axis versus Protein (mg per test tube or what we call the test volume) on the x-axis Note this is an AMOUNT of protein, NOT a specific concentration The objective is to calculate the CONCENTRATION of X and Y The highest number on the x-axis will be 3.0 This is obtained from the volume taken 0.6 mL (see protocol in the schedule) And the concentration of the standard protein solution, which is 5g/L 5g/L is equivalent to 5mg/mL (Shorthand for 5.0 milligrams per 1.0 mL) Therefore every 0.2 mL of standard protein solution contains 1mg of protein So 0.6 mL will contain 3.0 mg You can calculate all the other values by proportion From the best fit line drawn by hand you can read off the amount of protein in each replicate of X and Y from the x-axis For example it might be 1.6 mg protein per test volume From the protocol we see that 0.4 mL was taken of X and Y So to express the amount of the unknown as a specific CONCENTRATION In this case mg/mL (Shorthand for milligrams/ 1.0 mL) We need to multiply 1.6 by a factor of 2.5 (Because there are 2.5 lots of 0.4 mL in 1.0 mL) Thus the final concentration would be 1.6 x 2.5 = 4.0 mg/mL Do this for all 3 replicates of X and Y then calculate the mean and plug these values into the SEM calculation to estimate the standard error of the mean

8

Protein Assay calculations: Folin: The same procedure is applied for the Folin assay Except here the standard protein concentration is 0.2 mg/mL Thus 0.2 mL will contain 0.2 mg divided by 5 which equals 0.04 mg (Because 0.2 mL is one fifth of 1.0 mL) Therefore the largest volume taken to produce your graph (0.6 mL) Will represent 0.12 mg (3 lots of 0.04 mg) So 0.12 mg protein per test volume is the largest value on the x-axis Unknown samples X and Y have been diluted 20 times To convert X/20 and Y/20 to specific CONCENTRATIONS of mg/mL Read off the best fit line by hand to convert the Absorbance at 660nm to mg protein per test volume For example, it might be 0.08 Then we must multiply by a factor of 2.5 (1.0/ 0.4 because we took 0.4 mL of both X/20 and Y/20) And then multiply again by a factor of 20 Because for the Folin assay, which is more sensitive, but less accurate, the unknown samples X and Y have been diluted 20 times So 0.08 x 2.5 x 20 = 4.0 mg/mL Then do the stats The values of X and Y should be similar by both methods. If they are not how do you explain this? Hint: read the Introduction of the practical schedule.

9

Statistical Analysis Use your concentration values (mg/ml) of X and Y by both Biuret and Folin NOT your absorbance values.

1.

Calculate the mean Formula x 

2.

Example

x

(0.356  0.357  0.352) 3 xx  0.355mgml  1

xx 

n

Calculate the Standard Deviation Formula

Example x 

 (x 

x) 2

n

(0.356 0.355)2  (0.357 0.355)2  (0.352 0.355)2 3 0.000001 0.000004  0.000009 x  3  x  0.00216mgml 1

x 

3.

Calculate the Standard Error of the Mean (SEM) Formula  SEM  x n

Example SEM 

0.00216

3 SEM 0.0125mgml  1

10

Assessment Questions for Protein Assay by Biuret and Folin Methods 1.

The Biuret colour reagent interacts with which protein component?

A. B. C. D.

The N-terminus The C-terminus The peptide bonds The side chain of a specific amino acid

2.

The Folin colour reagent interacts with which protein component?

A. B. C. D.

The N-terminus The C-terminus The peptide bonds The side chain of a specific amino acid

3.

The standard protein solution provided for the Biuret assay is at a concentration of 5.0 g/dm3 which is the same as:

A. B. C. D.

5.0 mg/mL 0.5 g/mL 5.0  g/mL 0.5 mg/mL

4.

The standard protein solution provided for the Folin assay is at a concentration of 0.2 g/dm3 which is the same as:

A. B. C. D.

0.2 mg/mL 0.02 g/mL 0.2  g/L 0.02 mg/mL

5.

The numerical range on the X-Axis of your Biuret and Folin graphs are between:

A. B. C. D.

1.0 – 5.0 and 0.02 – 0.12, respectively 0.5 - 3.0 and 0.02 – 0.12, respectively 1.0 – 5.0 and 0.2 – 1.2, respectively 0.5 - 3.0 and 0.2 – 1.2, respectively

6.

The units on the X and Y – Axes of the Biuret assay graph are:

A. B. C. D.

Absorbance at 660nm and mg/test volume, respectively Absorbance at 660nm and mg/mL, respectively Absorbance at 550nm and mL/test volume, respectively Absorbance at 550nm and mg/test volume, respectively

11

7.

The units on the X and Y – Axes of the Folin assay graph are:

A. B. C. D.

Absorbance at 550nm and mL/test volume, respectively Absorbance at 660nm and mL/test volume, respectively Absorbance at 660nm and mg/test volume, respectively Absorbance at 550nm and mg/mL, respectively

8.

From your graph for the Biuret assay with respect to the unknown protein samples:

A. B. C. D.

X was present in a greater quantity than Y Y was present in a greater quantity than X X and Y were present in similar quantities, the values were fairly close There was no detectable protein in either sample X or Y

9.

From your graph for the Folin assay, with respect to the unknown protein samples

A. B. C. D.

There was no detectable protein in either sample X or Y X and Y were present in similar quantities, the values were fairly close X was ten times higher than Y Y was ten times higher than X

10.

From your calculated amount of protein for each of X and Y there should be correspondence between the two methods, but for one of the unknowns there isn’t and it gives an unexpected result. This is because the protein:

A. B. C. D.

Contains more tryptophan residues than would be considered normal Contains more tyrosine residues than would be considered normal Contains less tryptophan residues than would be considered normal Contains less tyrosine residues than would be considered normal

12

Name Student number

READ THESE INSTRUCTIONS CAREFULLY Circle the letter in the box that corresponds to the answer you think is correct. Note that there is only ONE correct answer for each question.

Question number

Answer Letter

1

A

B

C

D

2

A

B

C

D

3

A

B

C

D

4

A

B

C

D

5

A

B

C

D

6

A

B

C

D

7

A

B

C

D

8

A

B

C

D

9

A

B

C

D

10

A

B

C

D

Hand this grid in along with your tables, graphs, calculated protein amounts and SEM.

End

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