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[solved] Hunter College Bio 300 Lab Report#3 Protein Purification II Introduction: Beta galactosidase is an enzyme found in animals, plants and bacteria. It...


i have no idea where the purification table's numbers are coming from... (pages 11-12)?

also, can you look at this lab report and see if somethings wrong with it ??

also theres supposed to be 20 column fraction (page 7) but in this table there is 30 column fractions?? page 4.?




Hunter College

 

Bio 300

 

Lab Report#3

 

Protein Purification II

 

Introduction:

 

Beta galactosidase is an enzyme found in animals, plants and bacteria. It cleaves the ??

 

glycosidic linkage in disaccharide lactose to form its component monosaccharides, galactose and glucose. It is purified from the bacteria E.coli, which is made up of over 1000 amino acids. Protein purification is done in order to understand the sequence, structure and functions of a particular protein of interest. The first step in this process is the isolation of the protein of interest from all the other proteins in the cell. Then the protein is fractionated with the precipitation process using a salt such as Ammonium sulfate, which increases the ionic strength of the crude extract solution and makes the protein precipitate out. Once all the purifications steps are done, the protein then needs to be analyzed to check for purity. This could be done using the Lowry and the activity assay table and running calculations based on the Purification Table. Once purified, it is then possible to study the protein of interest.

 

The overall aim of this experiment was to perform purification of ??galactosidase and analysis of the purification. The aim of the first procedure in this experiment where the E.coli cell extract was created was to produce ??gal in an unregulated manner. The second procedure involved the precipitation with Ammonium Sulfate. This is done to obtain the ??gal in the supernatant and all other proteins in the resuspended pellet. The following procedure involved making a Lowry assay, which is done in order to calculate the total protein in the crude extract, the supernatant and the resuspended pellet. The last procedure was making ??gal activity assay, which was done in order to calculate the total ??gal activity in the crude extract, the supernatant and the resuspended pellet. The ratio of

 

these two gives the specific activity, which determines the purity of the fractions of our protein of interest (??gal).

 

The principle behind making the cell extract involved the addition of alumina to the cell pellet and grinding it in the mortar. This allowed the cell debris to be broken and allowed the proteins to come out. After centrifuging, the protein of interest went up to the supernatant and cell debris and other proteins went to the bottom as precipitate form. The ??gal activity assay is made by performing dilutions, which allows the unknown concentration of the enzyme to be within the range of the spectrophotometer. The ammonium sulfate precipitation was carried out with the principle of ?salting out?. The ?salting out? technique was based on the fact that the iconic strength of ammonium sulfate was sufficiently high enough to precipitate the protein out of the solution. The Lowry assay followed the principle of the reduction of the Folin?Ciocalteu reagent,

 

which gave rise the to the reaction of the copper ions with the peptide bonds in the protein. The total concentration of the protein in the sample was then deduced from the

 

residues of the Folin reagent. The ?-gal assay worked on the principle that there is

 

glycosidic bond in the disaccharide used in the experiment, which was hydrolyzed by ?gal. Specific activity is generally defined as the ratio of the unit of enzyme activity to the total amount of protein. In this experiment, we defined one unit of ??gal activity as the amount of enzyme required to produce 1 ?mole of PNP in one minute, from a pH 7, 6mM

 

PNP?Gal substrate solution incubated at 37oC. The total enzyme activity is the multiplication of the enzyme concentration with its volume. The total protein is the multiplication of the protein concentration with its volume. % Yield of protein is the total

 

protein in a fraction divided by the total protein in the starting material, then multiplied by 100. The specific activity determines the purity of the protein of interest. Method:

 

Preparation of E.coli cell extract: An overnight culture of E.coli strain was centrifuged and the pellet was collected. 10ml of pH 7.5 buffer was added to it and the pellet was broken up. The tube was centrifuged again and the pellet was kept in the freezer for a week. The pellet was slowly removed and its weight was calculated. It was then grinded in a mortar with alumina powder, which weighed 2 times its weight. Then 2.5 times the pellet weight gave us how much pH 7.5 buffer B was to be added to it. The mixture was then centrifuged and 1 ml of the supernatant was marked as the ?crude extract? and the volume of the rest of the supernatant was measured.

 

Precipitation with Ammonium Sulfate: The volume measured was multiplied with 0.291 to find out the amount of ammonium sulfate to be added to the crude extract. The mixture was then stirred for 30 minutes on a stir plate. It was centrifuged and the volume of the supernatant was measured. 1.8ml of pH 7.5 buffer was then added to the pellet and then swirled to get the resuspended pellet.

 

Lowry assay: 43 test tubes were filled using the Worksheet #1 and then 6.0ml of copper reagent was added to each tube. After 10 minutes, 0.3ml of Folin?s reagent was added to all the tubes. After 30 minutes, the absorbance at 500nm for each of the tubes was recorded. Spot test plate assay: 0.1ml of PNP was added to 33 wells of a microtiter plate. 0.01ml of each fraction was added to each of the wells and two other wells were filled with same amount of buffer B into one and resuspended pellet into another one. After 5 minutes, 0.05ml of 0.1N Sodium carbonate was added to each well and the wells that gave strong/weak yellow color, were recorded. ??galactosidase activity assay: 16 test tubes were filled with 3.0ml of PNP?Gal and were

 

put on a 37OC water bath. Then we followed Worksheet #2, and drew 0.05ml of D1 to tube #1. After 30 seconds, 0.05ml of D2 was added to tube #2 and the step was repeated for each tube. After 15 minutes, 3.0ml of 0.1N Sodium carbonate was added to each tube in the similar way. The absorbance at 405nm for each test tube was then recorded.

 

Experimental results: The Lowry assay showed a linear relationship between the Concentration of the protein and the Absorbances corresponding to the concentrations, which supports the Beer?

 

Lambert law.

 

Worksheet#1: Lowry assay

 

Test Sample

 

Sampl Volu

 

Absorpti Amount Fractio Fraction tube e me of on (nm)

 

of n protein #

 

volume buffer

 

protein protein amount

 

(ml)

 

(ml)

 

(mg/tub conc. (mgs)

 

e)

 

(mg/ml

 

)

 

1

 

Blank

 

0.0

 

1.2

 

0.00

 

0.00

 

0.00

 

0.00

 

2

 

0.1

 

1.1

 

0.125

 

0.06

 

3

 

0.1

 

1.1

 

0.120

 

0.06

 

4

 

0.2

 

1.0

 

0.200

 

0.12

 

5

 

0.2

 

1.0

 

0.210

 

0.12

 

0.6 mg/ml

 

6

 

0.4

 

0.8

 

0.390

 

0.24

 

Protein

 

7

 

0.4

 

0.8

 

0.420

 

0.24

 

(Standard 0.6

 

8

 

0.6

 

0.560

 

0.36

 

curve)

 

9

 

0.6

 

0.6

 

0.590

 

0.36

 

10

 

0.8

 

0.4

 

0.750

 

0.48

 

11

 

0.8

 

0.4

 

0.760

 

0.48

 

12

 

1.0

 

0.2

 

0.890

 

0.60

 

13

 

1.0

 

0.2

 

0.900

 

0.60

 

14

 

15

 

16

 

17

 

18

 

19

 

20 Column

 

Fraction #1

 

Column

 

Fraction #2

 

Column

 

Fraction #3

 

Column

 

Fraction #4

 

Column

 

Fraction #5

 

Column

 

Fraction #6

 

Column 0.2 1.0 0.05 0.015 0.075 0.0825 0.2 1.0 0.07 0.022 0.11 0.121 0.2 1.0 0.08 0.025 0.125 0.1375 0.2 1.0 0.10 0.042 0.21 0.231 0.2 1.0 0.11 0.044 0.220 0.242 0.2 1.0 0.09 0.04 0.20 0.22 0.2 1.0 0.06 0.02 0.10 0.11 21

 

22

 

23 Fraction #7

 

Column

 

Fraction #8

 

Column

 

Fraction #9

 

Column

 

Fraction

 

#10 0.2 1.0 0.075 0.024 0.12 0.132 0.2 1.0 0.08 0.025 0.125 0.1375 0.2 1.0 0.055 0.018 0.09 0.099 24 Column

 

Fraction

 

#11 0.2 1.0 0.06 0.02 0.10 0.11 25 Column

 

Fraction

 

#12 0.2 1.0 0.054 0.016 0.08 0.088 26 Column

 

Fraction

 

#13 0.2 1.0 0.07 0.022 0.11 0.121 27 Column

 

Fraction

 

#14 0.2 1.0 0.065 0.021 0.105 0.116 28 Column

 

Fraction

 

#15 0.2 1.0 0.059 0.019 0.095 0.105 29 Column

 

Fraction

 

#16 0.2 1.0 0.01 0.001 0.005 0.006 30 Column

 

Fraction

 

#17 0.2 1.0 0.069 0.022 0.11 0.121 31 Column

 

Fraction

 

#18 0.2 1.0 0.05 0.015 0.075 0.083 32 Column

 

Fraction

 

#19 0.2 1.0 0.065 0.021 0.105 0.115 33 Column

 

Fraction

 

#20 0.2 1.0 0.139 0.07 0.35 0.385 34 Column

 

Fraction

 

#21 0.2 1.0 0.250 0.128 0.64 0.704 35 Column

 

Fraction 0.2 1.0 0.03 0.001 0.005 0.006 #22

 

36 Column

 

Fraction

 

#23 0.2 1.0 0.07 0.022 0.11 0.121 37 Column

 

Fraction

 

#24 0.2 1.0 0.139 0.07 0.35 0.385 38 Column

 

Fraction

 

#25 0.2 1.0 0.03 0.001 0.005 0.006 39 Column

 

Fraction

 

#26 0.2 1.0 0.035 0.01 0.05 0.055 40 Column

 

Fraction

 

#27 0.2 1.0 0.061 0.02 0.10 0.11 41 Column

 

Fraction

 

#28 0.2 1.0 0.023 0.001 0.05 0.006 42 Column

 

Fraction

 

#29 0.2 1.0 0.032 0.01 0.05 0.055 43 Column

 

Fraction

 

#30 0.2 1.0 0.06 0.02 0.10 0.11 ALL FRACTIONS TOTAL PROTEIN= 4.307 mgs This table can be expressed as follows to create the standard curve for the Lowry assay.

 

Lowry Standard Curve For Ammonium Sulfate Fractions 1

 

0.9

 

0.8

 

0.7

 

0.6 Absorbance (nm) 0.5

 

0.4

 

0.3

 

0.2

 

0.1

 

0

 

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Concentrationof protein/tube (mg /tube)

 

Fig.1 Graph showing the relationship between the concentration and

 

absorbance for the Lowry assay

 

The ??galactosidase activity assay table below is drawn to get the dilution samples and their corresponding absorbances. This table follows the standard PNP curve for the following data.

 

??galactosidase activity assay: Test Sampl

 

tub e

 

e # Absorbanc

 

e (nm) ?moles

 

PNP forme

 

d Units Units/ml Dilution

 

(?moles (diluted) (inverse

 

PNP )

 

formed

 

per minute) Units/ml (undiluted

 

) 1 0.00 0.000 0.00000 0.00000 00.00 0.625 0.152 0.01 0.0005 10 0.005 0.140 0.035 0.002 0.0001 20 0.002 0.020 0.01 0.0006 0.00003 40 0.0012 0.005 0.002 0.0001 10 0.00005 0.005 0.002 0.0001 0.00000

 

5

 

0.00000 20 0.0001 2

 

3

 

4

 

5

 

6 Blank (buffer)

 

D19 1:10

 

D19 1:100

 

D19 1:1000

 

D20 1:10

 

D20 ?? 7

 

8

 

9

 

10

 

11

 

12

 

13

 

14

 

15

 

16

 

17 1:100

 

D20 1:1000

 

D21 1:10

 

D21 1:100

 

D21 1:1000

 

D22 1:10

 

D22 1:100

 

D22 1:1000

 

D23 1:10

 

D23 1:100

 

D24 1:10

 

D24 1:100 0.004 0.0019 0.0001 5

 

0.00000

 

5 40 >2.0 10 2.0 20 0.0002 0.215 0.051 0.0034 0.00017 40 0.0068 0.790 0.178 0.0119 0.00060 10 0.0060 0.145 0.032 0.0021 0.00011 20 0.0022 0.008 0.02 0.0013 0.00006

 

5 40 0.0026 1.85 10 0.189 0.046 .00031 0.00001

 

6 20 >2.0 0.0003 10 0.880 0.210 0.014 0.0007 20 0.014 PNP Standard Curve

 

1.4

 

1.2

 

1

 

0.8

 

Absorbance at 405nm 0.6

 

0.4

 

0.2

 

0

 

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Concentration umoles Fig.2 Graph showing the relationship between the concentration and

 

absorbance for the ??gal assay Elution Profile Fig.3 Graphical points showing the specific activities of the columns that gave positive signals. Here, open circles indicate the protein and the filled circles indicate the activity. Purification Table 1 (from last lab)

 

Item # Purification Step

 

Starting material:

 

Crude extract Fractions: Ammonium Sulfate ppt

 

Supernatant Resuspended pellet Vol (ml) 9.1 9.8 2.0 Protein conc. (mg/ml) 4.47 1.98 4.95 Total Protein (mg) 40.677 19.404 9.9 % Yield Protein 100% 47.7% 24.34% Enzyme activity (unit/ml) 21 0.272 112 Total activity (unit) 191.1 2.66 224 %Yield Activity 100% 1.39% 117.2% Specific Activity 4.70 0.14 22.63 Total Fold Purification ??????? 0.03 4.81 Discussion:

 

Based on the table of the Lowry and ??al activity assay, the data seem pretty consistent. According to the Beer?Lambert law, as the concentration of a sample is increased, its absorbance reading also increases and vice?versa. Both the Lowry standard curve and the dilution of the proteins supported the Beer?Lambert by showing the increase/decrease in the absorbance with the increase/decrease of the concentration of the protein respectively.

 

In the first purification, the volume of the filtered resuspended pellet was 1.15ml, and that

 

of the most pure fraction was 0.1ml. The concentration of the filtered resuspended pellet was 4.95 mg/ml and that of the most pure fraction was 0.125mg/ml. The total protein in the filtered resuspended pellet was 5.14 mg, all fractions were 4.307mgs and most pure fraction was 0.125mg. From the entire filtered resuspended pellet, 83.79% protein ended up in all fractions and only about 2.43% in the most pure fraction. However, the addition of them does not add up to the 100% of the starting crude extract. The reason for this discrepancy could be because some of the proteins of interest may have denatured somewhere during the process because of so many steps involving in its purification. The

 

concentration of the filtered resuspended pellet for the ??gal activity was 112 unit/ml, and

 

that of the most pure fraction was 0.00036. The total amount of ??gal activity in the filtered resuspended pellet was 128.8 units, in all the fractions were 0.0033 units, and in the most pure fraction were 0.00003 units. Only about 0.002% of the ??gal activity was found in all the fractions and only about 0.00002% of it was found in the most pure. One reason for this discrepancy could be because the absorbances were recorded wrong, causing interruptions in the calculations. Also, initially the volume of the supernatant should have been less than that of the crude extract. This misreading might have led to any discrepancy found in the latter calculations. In the second purification, the volume of the cell extract was 9.1ml, and that of the resuspended pellet and most pure fraction were 2.0ml and 0.1ml respectively. The concentration of the cell extract was 4.47 mg/ml, that of the resuspended pellet was 4.95 mg/ml and that of the most pure fraction was 0.005mg/ml. The total protein in the crude extract was 40.677 mgs, that in the resuspended pellet was 19.404 mgs and that in most pure fraction was 0.125mgs. From the entire filtered resuspended pellet, 47.70% protein ended up in the resuspended pellet and only about 3.07% in the most pure fraction. However, the addition of them does not add up to the 100% of the starting crude extract. The reason for this discrepancy could be because some of the proteins of interest may have denatured somewhere during the process because of so many steps involving in its purification. The concentration of the crude extract for the ??gal activity was 21 unit/ml, and that of the resuspended pellet and the most pure fraction were 112 unit/ml and 0.00036unit/ml respectively. The total amount of ??gal activity in the crude extract was 191.1 units, and that in the filtered resuspended pellet and the most pure fraction were 224 units and 0.00003 units respectively. About 117.2% of the ??gal activity was found in the resuspended pellet and only about 0.000016% of it was found in the most pure fraction. The two % add up to more than 100%, which is ambiguous. According to the Purification Table, it is seen that the specific activity of the most pure fraction was much lower and that of the resuspended pellet was much higher than the specific activity of the crude extract. The total fold purification in the resuspended pellet was 2.46 and that in the most pure fraction was 0.00005. We have learnt that high purity corresponds to the low yield of the sample. Here, most of the pure ??gal activity was in the resuspended pellet. So I think the second purification was not really necessary, because that didn?t significantly change the results. The cons of the two techniques are that, it requires too much work and time, also, a lot of money is wasted in the production of the pure ??gal.

 


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