Experimental Protein Extraction And Separation Essay, Research Paper

ABSTRACT

Proteins are the macromolecules that are responsible for most of the bodily functions. By investigating an individual protein, one can be able to understand the functions and structure of an organism. Before this can be done, protein has to be separated from cell components. Using the methods of centrifugation and gel electrophoresis, not only a protein can be separated from cellular components but also from other proteins. An experiment was designed to separate protein contained in liver tissue of a cow based on it’s molecular weight. From the centrifugation, soluble protein concentration extracted for the electrophoresis. Dilution was prepared of this concentrated extract; positive correlation made between the concentration of BSA and measured absorbance. This correlation helped to draft a regression line, which was useful in finding concentration of protein extract. An electrotransfer on PVDF membrane was done after the gel electrophoresis. These migrations of proteins based on their molecular weight of three solutions. Using the polyacrilamide electrophoresis, proteins were separated based on their molecular weight. Extraction buffer did not produce protein bands. Proteins with huge molecular weight did not travel far from the gel while low molecular weight proteins traveled long distance . Overall banding contrast was compared between same sample with different transferred volumes. The protein bands of ADH were closely related to some of the bands of liver protein extract. ADH, which is derived from horse contains some of the protein that are related to the proteins found on cow’s liver cells. Closely ranged Rf value is key to similarities that exists between protein bands based on their molecular weight. The results of these experiments were compared with research group with similar interests; the results and conclusion drawn were closely correlated.

Introduction

Our body consists of various types of macromolecules, which are the fundamental functional unit of life. These macromolecules are responsible for most of all of the cell functions. They include molecules such as carbohydrates (glycogen, starch, cellulose, chitin), lipids (fats, fatty acids, steroids, phospholipids), nucleic acids (RNA, DNA, rRNA, mRNA, tRNA), and proteins (1,2).

These proteins are the molecules that regulate all of the bodily functions in an organism. Proteins provide structural, chemical, hormonal, and metabolic support an organism attain for everyday life. Proteins acts as transporters to synthesize genes, regulatory functions as found in membranes, receptors to specific ligands, and structural support found in muscles. All proteins are made from the monomers of amino acids (1,2).

The chain of amino acids is linked by a peptide bond; peptide bond in synthesized by peptidyl tranferace located in the large ribosomal unit of the ribosome. The shape or conformation determines the function of a protein. The shape is determined as protein’s polypeptide chain is synthesized by the ribosome (1,2).

The structure of proteins is separated into four organizational level (primary, secondary, tertiary, quaternary); it is based on the types of interactions among individual amino acids on a protein. These interactions causes bonds (ionic, hydrophobic, disulfide, hydrogen) to form, which gives protein its conformation. The bonds occur between the R-group, C-terminal end, and N-terminal end of amino acids in single or more polypeptide chains of a protein(1,2). Proteins are microscopic molecules that consists of various molecular weight, shape, and charge. One cannot study the functions and structural variations in an intact cells(2).

The protein must be separated using proper methods and techniques in able observe and calculate special properties. Proteins further can be targeted using antibody (6) The separated proteins are used as procedural method to purify a particular protein. The techniques used for protein separation is important in able to further investigate the native structure, and functions of a protein (2). The procedures of protein separation is a trivial procedure for laboratory workers.

Prior to protein separation, the intact cell and other cellular materials needs to be removed from the molecules of interest. By homogenization, cell can easily be turned into cellular debris except for brain cells, which contains abundant amount of myelin. For brain cells a mechanical homogenizer is used for cell debris (3,6). The hand held homogenizer is used for most cells except for the brain cells.

After intact cells are crushed and turned into cell debris, the suspension is ready for centrifugation to obtain molecules of special interest. Cellular suspension is separated based on the mass against force of gravity. After centrifugation, protein extract is ready for gel electrophoresis. Protein separation is proficiently done using gel electrophoresis (4). In recent years, a better techniques of electrophoresis are being used for protein separation. These techniques are integration of gel electrophoresis and capillary electrophoresis (5).

An experiment was performed using techniques of centrifugation, and gel-electrophoresis to obtain and separate proteins found in cow’s liver cells. The liver cells acquire large amount of stored proteins and carbohydrates body needs daily functions. ADH was used as a comparison protein markers against the extracted soluble proteins under gel electrophoresis. A specific protocol and procedure was developed in able to separate protein molecules found in cow’s liver cells based on their molecular weight as described in Brand (7).

EXPERIMENTAL METHODS

Extraction of cow liver cell proteins

A liver tissue from Bos taurus (cow) was provided by University of Texas laboratory preparation division. 1.92 g of liver tissue was chopped into fine small pieces by a razor blade and added to chilled mortar and pestle; extraction buffer of 5 ml containing 50mM PIPES, 1mM MgCl2, 1 mM DTT, 10 mM DIECA, 10 mM Leupeptin, and 10 mM Pepstatin at pH 6.9 was added into the mortar. The grinded liver tissue was homogenized using the pre-chilled homogenizer submerged in ice. The liver tissue was crushed until the containing extraction buffer became homogeneous (7). Afterward, cellular liver contents in the 5 ml extraction buffer was centrifuged at maximum speed for 15 min in a refrigerated clinical centrifuge. The collected soluble cell extract was stored in bottle and cooled in ice (7).

The parallel dilution method was applied to obtain six 0.5 ml dilution of 1.0 mg/ml, 0.5 mg/ml, 0.3 mg/ml, 0.2 mg/ ml, 0.1 mg/ml, and 0.05 mg/ml using bovine serum albumin (BSA) standard A4503 (Sigma stock solution containing 2.0 mg protein/ml) as described in Brand (7). The six dilution are placed in separate Spectronic cuvettes. 4.9 ml of Bradford’s reagent 500-0006 (BioRad, working solution 1:10 dilution) was added to these six dilution The collected protein extract was also diluted with dH2O in ratios of 1:30, 1:50, and 1:100. 0.1 ml of each dilution and 4.9 ml of Bradford’s reagent was added to the Spectronic 20 cuvettes; absorbance of all the Spectronic 20 cuvettes was taken at 595 nm (A595).

Using mentioned extraction buffer, protein extract dilution was prepared at ratio of 1:30; 150 ml of 1:20 protein extract and extraction buffer were poured in separate eppendorf tubes.

50 ml of 4X sample treatment buffer (containing SDS detergent, 2-mercaptoethanol, glycerol, 0.3 M Tris, 10% w/v SDS, 1.25 mg/ml Bromophenol blue) was added to each into prepared tubes (6). The tubes were incubated in 60.C water bath for 15 min with manual agitation every 5 min. The tubes were stored in freezer (7).

Gel electrophoresis of extracted proteins

The small slab gel unit mighty SE200 series (Hoefer) containing 3% polyacrilamide as stacking gel and 11% as separating gel; gel was separated into 10 wells for electrophoresis. Previously stored eppendorf tubes and tube containing alcohol dehydrogenase (ADH) from University of Texas preparation of laboratory division were bathed in 60.C water for 3 min. Tank buffer was added after slab gel unit was attached to electrophoresis apparatus (7). 5 ml of SDS-PAGE Standard Molecular Weight Markers Proteins broad-range #161-0318 (BioRad) in well #1 and #6, 10 ml of protein extract in well #2 and #6, 10 ml of extraction buffer in well #3 and #8, 15 ml of protein extract in well #4 and #8, and 10 ml of ADH was inserted in well of slab gel unit accordance to guidelines (7,8,4). A 33mA of electric power was applied to gel using BioRad PowerPac 300 for 28 min duration.

First five lanes were sectioned from rest of the gel; it was treated into gel staining solution containing 0.125% w/v Coomassie Brilliant Blue R-250, 50% methanol and 7% acetic acid. This petri dish was agitated using Labline Reciprocol Shaker for 45 min. The gel was rinsed with dH2O and placed for 15 min in Fresh Destaining Solution (containing 50% methanol, and 10% acetic acid) on Labline Reciprocol Shaker. Two more intervals of 15 min containing half full Fresh Destaining Solution and folded laboratory tissue wiper were added to the gel. The gel is stored as described method in Brand (7).

Protein electrotransfer

The second half of gel was placed in a different petri dish containing cathode buffer. This gel was subject to protein electrotransfer for 30 min using PVDF membrane P0682 (Sigma) and MilliBlot electrotransfer unit (Millipore) as described methods in Brand and Farrell (7,6). The gel was stained for 2 min using Coomassie Brilliant Blue. The PVDF membrane was placed for 5 min in solution containing Tris buffer and NaCl; the PVDF membrane was placed on Labline Reciprocol Shaker for 2 min after PVDF membrane was placed in the same petri dish as gel.

The membrane was placed into half full Fresh Destaining Solution ; the membrane was agitated using Labline Reciprocol Shaker for 5 min. Petri dish containing gel in Coomassie Brilliant Blue was placed back on Labline Reciprocol Shaker for 20 min. The membrane was stored for 1 week as described in Brand and Farrell (7,6). The gel was destained two more 15 min intervals using Fresh Destaining Solution. After destaining was performed, gel was discarded.

Characterization of separated proteins from liver

Stored gel was gel was placed on clear plastic wrap and over a 8.5 X 11 in. white paper. The protein bands’ distance in Standard Molecular Weight Markers Protein (Lane #1) and ADH (Lane #5) were measured from their origin. Of all the protein bands in 15 ml protein extract (Lane #4), only few distinctly seen protein bands’ distance were measured.

RESULTS

The centrifugation and gel electrophoresis techniques were used in an experiment to separate different types of protein found in liver cells of a cow. The protein extract obtained was compared to alcohol dehydrogenase taken from a horse. The protein were separated in gel electrophoresis based on their molecular weight.

Extraction of cow liver proteins

From the 1.92 g of liver tissue that was chopped to produce supernatant containing 6 ml of soluble protein. The recorded absorbance values (Fig. 1) were used to determine the protein extract concentration (Fig. 1a) before and after the dilution (Fig. 2). The BSA stock solution used contained 2.0 mg protein per ml.

Figure 1: Dilution of BSA

BSA Conc. (mg/ml) BSA Volume (ml) A595

0.05 12.5 0.08

0.1 25 0.16

0.2 50 0.27

0.3 75 0.34

0.5 125 0.52

1.0 250 0.54

In able to know the known concentration of soluble protein extracted. A standard curve (Fig. 1a) was developed to measure the concentrations protein extract after the dilution, which also provides to know the initial concentrations of protein extract at various volumes (Fig 2).

21.5 mg/ml is the actual concentration of protein extract before the dilution was made using dH2O. 129 mg was the amount of soluble protein obtained from 6 ml of supernatant. 6.71% of the 1.2 gm of liver tissue is obtained as a protein extract to create a ratio of 67.18 mg of soluble protein per gm of liver tissue. Using the dilution, protein extract decreased in protein concentration (Fig. 2) per ml.

Figure 2: Dilution of Liver Protein Extract

Volume Extract in the cuvette A595 Protein Conc. (mg/ml)

(595 nm) After dilution Before dilution

5 ml 0.62 0.615 18.45

2 ml 0.51 0.491 24.55

2 ml 0.46 0.440 44.00

Electrophoresis

Gel electrophoresis was performed on the protein extract, ADH, protein markers, and extraction buffer using the sample treatment buffer. Tank buffer was used to fill up the reservoirs of the electrophoresis apparatus. Slab gel unit contained 10 wells; slab unit was equally divided. One half of the gel was used for method of electrotransfer on PVDF membrane.

After 33 mA of power applied to the gel, it took 28 min for the sample treatment buffer to migrate through the gel. No proteins are visible at this stage; the Coomassie blue quickly stained the gel with blue dye. All the protein bands contained color from the blue dye.

ADH and molecular weight marker proteins produced fewer protein bands. ADH produced only three bands while molecular weight protein markers produced seven distinct bands. Both protein extract lanes at different volume produced several (>35) bands. Proteins bands of 15 ml protein extract were much dense and distinct then the 10 ml extract. Lane #3 (extraction buffer) was completely blank. No protein bands were noticed on Lane #3.

Protein electrotransfer

15 rectangular pieces of filter paper were cut in size of 5.5 X 4.5 cm. The filter paper were stacked above and below the gel, and PVDF membrane. MilliBlot electrotransfer unit was used for 30 min. Unmeasured volume of cathode buffer was used to soak the gel and the PVDF membrane. The PVDF membrane showed protein bands more distinctly than the gel itself; PVDF was easier to handle than the gel.

Measuring the Rf values

Rf, the rate of migration of proteins based on their molecular weight, was measured for the molecular weight protein markers, ADH, and the protein extract. Rf value for protein extract bands were taken from the 15 ml extract lane. 5.4 cm was measured as the gel front.

The molecular weight marker protein were characterized based on their length from the origin. Therefore, protein bands of different protein were identified using the reference provided by BioRad (Hercules, CA)(Fig. 3). The Rf values of each protein separated was calculated from the measured distances from the origin (Fig. 3). BioRad (Hercules, CA) also provided the molecular weight (kD) of the separated proteins (Fig. 3).

One of the protein (aprotinin) has a very small molecular weight (7 kD)(Fig. 3). It’s protein band was not noticed after the electrophoresis. Some of the bands in Lane #1 (molecular weight marker protein) were not clearly distinct.

Fig. 3: Characterization of Protein Molecular Weight Markers

ADH was also measured in values of Rf (Fig. 4). Only three bands of ADH proteins were noticed. All three bands were found in the upper half of the gel. The length of the gel front remained the same at 5.4 cm.

Figure 4: Rf in ADH

Rf values of the liver protein extract were also calculated (Fig. 5). There were morethan 15 protein bands separated by electrophoresis in Lane #2 and #4. Lane #4 (15 ml protein extract) was chosen to measure bands from the origin. The bands were distributed from the origin all the way close to the gel front. Many of the bands were too close to one another.

Figure 5: Characterization of Liver Protein Extract

In Fig. 5, the molecular weight (kd) of the randomly chosen protein bands is also calculated using ploted semi-log graph (fig. 3a). Since the ADH was the protein marker, it can be used to base other protein bands. ADH serves as a reference guide for the extracted proteins. The 13 measured values in Fig. 5 were randomly chosen from the bands present in the Lane #4. 15 ml used to show these protein bands were more distinct and dense than the bands found in 10 ml protein extract (Lane #2).

CONCLUSIONS

The overall period of the experiment took 3 week period. The performed experiments were done at University of Texas at Austin Biological Science department. Soluble protein of liver tissue was collected using centrifuge techniques. The protein extract was compared in gel electrophoresis with ADH derived from horse. The proteins were successfully separated based on their molecular weight (kD).

There is a positive correlation between the BSA volume added and the absorbance at 595 nm ( Fig. 1). A regression line can be drawn from the plotting (Fig. 1a) to measure the concentration of protein extract at any absorbance value. Using the Spectronic 20, absorbance value of the protein extract was measured (Fig. 2); the absorbance values at various concentrations helped to determine concentrations of protein extract before and after the dilution by using a regression line. Before the dilution was made, the protein extract was extremely concentrated (Fig 2); dilution with dH2O helped the experiment to obtain better data.

The parallel dilution method was used for above dilution (7). BSA extract used was carefully and completely purified. This helped to present valid data points. Since, the data in Fig.2 is partially depended on the BSA concentration vs. Absorbance. The first dilution of protein extract was invalid; mistakes were made using the equipment. These mistakes include: automatic pipette was used incorrectly, and setting the correct volume on the pipette. These two errors were fixed soon as the mistakes were found. After these errors, the automatic pipette was used correctly according to guidelines provided by laboratory personnel. The data was compared for the first part of the experiment with other research groups. A positive correlation was made among the research groups

The gel electrophoresis of the protein extract, extraction buffer, and ADH was done. The second part of the experiment went smoothly without any mistakes. The proper guidelines provided by laboratory personnel and in Brand (7) was followed. Protein bands observed in Lane #5 were not perfectly horizontal, instead, protein bands were in “wavy” like configuration. This made it difficult to measure the distance of each bands from the origin (Fig. 4). Several protein bands were noticed in the lanes containing protein extract. Lane #3 did not produced any protein bands. The extraction buffer did not contain any proteins; extraction buffer served as the controlled experiment. Since, extraction buffer was mixed with protein extract.

The gel electrophoresis done was compared to other research groups. In comparison, other group’s gel was not completely destained. Their gel for that reason did not produce distinct and attractive patterns of protein bands. To produce distinct contrast of protein bands, the gel was destained many more times using the tissue paper and destaining solution. More time the destaining solution was used, protein bands became more distinct against the gel background.

The protein electrotransfer was prepared on the other half of the slab gel unit. The MilliBlot electrotransfer unit was used to transfer the protein bands from the gel to the PVDF membrane. Protein would precipitate from the gel to the PVDF membrane based on electrical charge distribution. Cathode buffer was used to soak both the electrotransfer membrane and the corresponding gel. Cathode buffer used was not measured. Proper procedures were followed during this part of the experiment. The electrotransfer membrane and gel were not correctly placed in MilliBlot unit. This caused ineffective contrast of protein bands on the membrane. The protein bands on the membrane were not clearly distinct; they faded into the membrane background. Other groups’ membrane produced bands that were clearly noticeable.

Protein bands were compared among lanes. Looking at Fig. 3, 4 and 5, protein bands distance from their origin can be compared. ADH protein bands were found closely related to the bands in Lane #2 and #4 (protein extract). This can suggest that some of the protein found in horse and cow have similarity based on their molecular weight. For this reason, one can use the Fig. 3a in able to approximate the molecular weight of the proteins separated from the protein extract. Just because the bands weigh the same does not make them similar in molecular and structure composition. Other groups’ Rf values for lane #1 and #5 were similar. It was difficult to figure exactly where on the protein band to measure the distance from the origin. Since each protein bands were only few mm long, the measurement of each protein band was take from the center of that band to the origin.

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