Protein Solubilization Visualization And Identification Using Various Technique Biology Essay

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Abstract:

The aim of this study was to characterize the effects of different solvent on protein solubilization visualized by analytical techniques including Bradford assay vs Dumbroff/Ghosh assay, SDS-PAGE, coomassie blue and silver staining techniques. Protein was identified using western blotting and mass spectrometry. Results indicate that PBS+2% SDS is the better solvent because it showed the highest protein concentration and coomassie blue is a better staining technique instead of silver staining for large amounts of protein. Mass spectroscopy showed better and more accurate protein identification than western blotting.

Introduction:

Proteins are biochemical macromolecules that are essential for almost every bodily function. They are formed when carboxyl group of one amino acid is linked with the amino group of another amino acid forming a peptide bond. Proteins can either be present alone or in large complexes that are joined by disulfide bonds, week electrostatic forces or can be polymerized into huge insoluble complexes (1). Proteins can form large complexes with varying charges which are often difficult to separate. Thus, in order to determine the amount of protein present in a sample it is important to perform protein solubilisation (1). Transforming non-soluble forms of proteins into soluble form is called protein solubilisation. Proteins are solvent specific, which mean every protein solubilises in a specific solvent. Some solvents that can solubilize proteins include EtoH, water, PBS+SDS, PBS+Triton, and PBS etc.

Use of solvent and their effect on protein solubility:

Some of large proteins easily solubilize in organic solvents such as ethanol or acetone, whereas others are able to solubilize in water. Some require addition of salt in water, and some are sparingly soluble in salt water and require the use of detergents (2). Detergents play crucial role in manipulation, purification and extraction of membrane proteins. They are amphipathic in nature i.e. they possess both hydrophilic and hydrophobic characteristics. They can imitate the natural environmental of lipid bilayer and solubilize the protein. There are two types of detergents ionic and non-ionic (2).

PBS:

Phosphate Buffered Saline (PBS) is a non-toxic isotonic buffer and water based salt solution. It is mostly used as diluent which dries bio-molecules such as proteins. It is a mixture of sodium-potassium phosphate and sodium-potassium chloride with a pH of 7.4 (3).

PBS+2% SDS:

Sodium dodecyl sulfate (SDS), is an anionic detergent that consists of a charged head group (+/-) with a long hydrocarbon chain and a water soluble ionic group. SDS is able to lyse the cell and disrupts hydrophobic interactions and non-covalent bonds by providing a hydrophobic environment, which results in the dispersion of monomeric moieties in the environment and making these residues negatively charged (4). Membrane proteins can be readily solubilized by ionic detergents.

PBS+ 1% Triton X-100:

Trition X-100 is a non-ionic detergent that can also be used for protein solubilization. Unlikely, SDS it consists of an uncharged hydrophilic, either glycosidic groups or polyoxyethylene head group (5). These types of non-ionic detergents isolate proteins from lipid in their active form i.e. without disturbing the protein structure, because they break lipid-protein and lipid-lipid interactions instead of protein-protein interactions (5).

Water and ice-cold 100% Ethanol:

Proteins are readily soluble in water because it is a polar solvent with a pH 7. It has been studied that when water is used as a solvent it increases proteins' solubility twenty- folds. On the other hand, different proteins require different concentration of ethanol to be solubilized. It has been shown that ethanol lowers the protein solubility because in the presence of ethanol, proteins soon after unfolding themselves re-fold into rod -like structures with exposing α-helices (6). Proteins tends to bury the some proteins are not stable in solvents like ethanol (1).

Protein solubility depends on many factors such as charge, pH and nature of solvent such as its polarity. Protein consists of a net charge which is determined by the nature of amino group present and as well as on the value of pH that turns zero at iso-electric points (PI) (7). It is important to know that solubility minimizes when pH reaches iso-electric point. Furthermore, folding of the protein structure is also stabilized by many factors such as when more than 80% of the peptides and non-polar side chains are buried and tightly packaged; it contributes toward the protein stability (7).

An analytical technique to measure the concentration of protein present in a solution is called protein assay. Bradford protein assay is one of the most employed techniques in determining protein concentration. It has both its advantages and disadvantages. Bradford assay is quite accurate and fast, which is used to determine the concentration of protein before performing gel electrophoresis (8). The basic phenomenon behind this procedure is that it involves binding of coomassie dye with the protein (8).

Coomassie dye has four different ionic forms. But only one anionic blue form binds with the protein present in the solution. The blue anionic form i.e. coomassie brilliant blue G-250 dye binds with the protein present in the solution and has an absorption maximum in a range from 590-595 nm (4). Thus, assessing the amount of dye which binds with protein yields the concentration of protein present in a solution. Coomassie blue usually binds to arginyl and lysyl amino acid residues of protein (4).

There are some disadvantages of Bradford method, presence of the compounds like detergents, such as Triton-X100 or SDS hinders the binding activity of the dye with the protein. Also, coomassie dye is size specific. It has been shown that it can only binds with proteins smaller than 3000 Da (4).

Dumbroff/Gosh protein assay is another useful method that can be applied to determine the protein concentration present in the solution. It is much accurate and fast when vast numbers of sample are processed. This method make use of a whatmann filter paper, on which sample is spotted and let dried. The filter paper is covered with coomassie blue dye and rinsed with 20% MeOH(acidified), which then allows to read the protein concentration as a reflectance using a scanning densitometer (9). Unlike to Bradford protein assay, this method is more accurate as well as it does not get hindered by the presence of any detergent, thus produces better outcomes. It is more sensitive than Bradford method and can be reused over several months because it shows long term stability for protein-dye complex (9). Protein concentration found using Bradford protein assay and Dumbroff/Gosh protein assay method for this study was 4.98mg/ml and 5.00mg/ml, respectively.

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) is a bio-analytical technique that helps in the separation of proteins from their native structure. Protein separation is based on electrophoretic mobility. SDS-PAGE helps to separate proteins by giving them identical charge-to- mass ratio, i.e. negative charge on all protein, thus making molecular weight the only criteria for protein separation (7). The gel is made of polyacrylamide; ammonium per sulfate (APS) is used as an oxidising agent, and Tetramethylethylenediamine (TEMED) is used as a catalyst. The gel separates protein based on their molecular weight and not on their native charge, which means that protein with higher molecular weight stick at the top of the gel, where as proteins with smaller molecular weight migrate faster towards the bottom of the gel (10). In order to achieve the best results, reducing agents such as (Dithiothreitol) DTT are applied that breaks the covalent bond or the di-sulfide bonds between the amino residues (10).

APS helps to commence polymerization, whereas TEMED polymerises the gel very quickly. APS and TEMED is responsible for the rate at which polymerization occur and resultant properties of the gel such as turbidity, elasticity etc. For example, if the concentration of TEMED and APS is increased, the gel elasticity would decrease because the average chain length would decrease, thus increasing the gel turbidity (10).

To visualize bands that may have appeared on gel after electrophoresis is accomplished by staining the protein with dye. Coomassie brilliant blue is usually considered for this process because of its sensitivity, clear background, good compatibility with MS and reproducibility, these volatiles the silver staining techniques (11). Coomassie family is insensitive to parameters such as temperature, time required for development and quality of solvent. Its lower sensitivity is balanced with its reproducibility. Coomassie blue stain possesses staining intensity which is due to the dye-protein interaction, in which dye is hydrophobically bonded to the protein residues (11). Presence of Lys and Arg residues on polypeptides favours the binding of coomassie dye with the protein molecule (4).

Silver staining technique helps to detect biological compounds such as proteins that may be present on solid medium like gel. The presence of these biological compounds produces brown band which become very visible. Silver staining technique is 10-100 time more sensitive when compared with coomassie blue staining technique. However silver staining shows poor compatibility with mass spectrometry (MS) because of the presence of glutaraldehyde, which is able to cross-link with polypeptide chains (12). Silver staining works on the reduction reaction. Once gel is preserved with soluble silver ions it is developed by exposing to the reducing agent such as formaldehyde. Gel reduces silver to the elemental silver giving off brown precipitate. This technique has an advantage over coomassie staining, because presence of sodium thiosulfate blocks the silver ions in the solution thus inhibiting the staining of the unwanted background (12).

Western blotting is a technique employed to detect a protein of interest from a crude extract. This method involves, transferring of a protein on to a nitrocellulose membrane or polyninylidene difluoride (PVDF) filter through electrophoresis (13). This helps to expose proteins because PVDF and protein make non-covalent bonds. Nitrocellulose membrane is treated with methanol to activate it before exposing it to antibodies. The membrane-protein complex is exposed to the primary antibody which tightly binds with specific proteins on the membrane. In the presence of primary antibody, another antibody called secondary antibody, labelled with radioactive substances such as 14C , 3H etc also known as anti-mouse is applied (7). Secondary antibody consists of a marker called reporter enzyme that is covalently attached to the antibody and helps in the visualization of the target proteins. Secondary antibody undergoes a chemi-luminescent reaction when it binds to the target protein, thus giving off a visible color change (7). Some common primary antibodies include α-beta-actin, α-GADPH and α-BSA. Furthermore, secondary antibodies are produced when host animals are immunized against immunoglobin proteins. Goat alpha mouse IgG H+L is an example of secondary antibody that binds with primary antibodies. GADPH is a monoclonal antibody that produces a single clean band when binds with the protein of interest in western blotting technique. A α-beta-actin, is also an antibody that interact with broad range of species such as human or cow etc (13).

Protein autoradiography is a technique employed to visualize proteins. This technique detects proteins with the help of photogenic emulsion. In order to do emulsion, X-ray films are used. Dark bands appear on the gel when the film is developed in the dark room because these bands produce radioactive emissions that are exposed to the film (7).

Binding of antibody is a very crucial step because there is greater chance of antibodies binding with the membrane due to the high affinity of proteins. In order to avoid this risk, measures should be taken. For example, it has been studied that, before exposing the membrane to the antibody it should be diluted with protein solution which blocks this from happening. Bovine serum albumin (BSA), is one of the blocking solution that is mostly employed for this procedure. Non-fat dry milk can also be used with minor concentration of a detergent (14). This step allows the protein in the blocking solution to be attached with the membrane, thus leaving no room for the antibodies to bind. Therefore, when antibodies are added over the membrane, they bind with the target protein instead of the membrane (14).

To obtain better results it is important to reduce the background and remove any unnecessary bounded reagents. In order to accomplish this washing buffers are used such as PBS. They increase the noise: signal ratio, thus improving the results.

In order to characterized and find out mass of the protein, a technique called protein mass spectrometry is employed. It consists of the two primary method for the ionization of the protein i.e. electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). ESI method produces ions through ionization of small amount of large macromolecules such as proteins. It works on the atmospheric pressure where macromolecules are bombarded with an energetic beam of electron, resulting in the formation of ions (15). The fragmentation of ions produces large number of cations and radicals. Protein fragmentation involves mixing of the sorted peptides with the molecular ions, thus producing peptides by cleavage. A sample is sprayed to the strong magnetic field to measure the masses of the ions. Lighter ions will be deflected more than the higher ions when passed through the magnetic field, thus this helps to determine the mass to charge ratio (15). Mass of the molecular ions provide the information of the molecular mass of the sample, where as mass of the fragment ion provides information on the compound structure (15).

Similarly, MALDI method is a more sensitive soft ionization technique, which allows the analysis of bio-molecules such as proteins and organic molecules. These two methods for protein characterizing are refer as top-down methods (15). Another method called bottom-up method is also considered for protein characterizing. In this approach smaller peptide are formed, when proteins are digested with the help of protease enzyme such as trypsin. Trypsin cleaves the peptide chain at lysine and arginine (16). However, it is interesting to note that when proline is present at the end of the peptide chain, then protein is ionized in various length of peptides.Smaller peptides are easier to load on mass spectrometer and one such method to load peptides on mass spectrometer is called electrospray ionization (16).

The purpose of this experiment was to identify the effects of different solvents protein solubilization which was accomplished through Bradford assay vs Dumbroff/ Gosh assay, SDS-PAGE and coomassie vs silver staining technique.

Materials and Methods:

~0.1g of Bovine liver protein was obtained and homogenized with either of the solvent i.e. 100% ice-cold ethanol, PBS + 1% Triton X-100, PBS +2% SDS, water, and PBS. Homogenate was transferred to a Dounce homogenizer, and homogenization was carefully carried out with 10 strokes of loose pestle and 10 strokes with tight pestle. The homeogenate was then transferred to 15 mL tube and stored at -20°C. Bradford and Dumbroff/Gosh protein assay were performed to determine the protein content in the solution. For Bradford assay dilution of each sample i.e. 1:1, 1:10, 1:150 were prepared using 1Ã- PBS. Protein dilutions were made using 100μL of the dilution, 4.5mL of 1Ã- PBS and 500 μL Bradford reagent. Color reaction developed after 5 minutes. Transmittance was read at 595nm. For Dumbroff/Gosh assay dilutions were made using 2% SDS i.e. 1:1, 1:10, 1:150. 2 μL of each dilutions and BSA standards were pipette on Whatmann #1 filter paper.Filter paper was then stained with coomassie blue for five minutes until royal blue color appears. Paper was then destained until all dye was removed and allowed to dry and then scanned.

Protein sample was then subjected to SDS-PAGE gel electrophoresis. 9% separating gel was prepared by 2.25mL 30% acryl/0.8%bis, 2.25mL 3.0M Tris-HCL+0.3%SDS, 2.25mL UF water and 0.75mL glycerol. 10 μL TEMED and 25 μL 20% APS was added to the gel. The gel was allowed to polymerize for 5 minutes at room temperature. 4% stacking gel was prepared by adding 0.82mL 30% acryl/0.8%bis, 1.55mL 3.0M Tris-HCL+0.3% SDS and 3.89mL UF water. 10 μL TEMED and 25 μL 20% APS was added to the gel. Teflon comb was inserted into the layer of stacking gel. 5μL protein solutions were loaded in lane 2-5 and 7-10 and 5μL molecular weight marker " Froggabio- Tri-glycine 4-20% was loaded in lane 1&6 on the gel.

Western blotting was demonstrated to identify the presence of protein of interest. The primary antibodies used were α-beta-actin, α-GADPH and α-β-BSA and α-BSA. The secondary antibodies used were Goat-α-Mouse IgG and H+L HRP. 9% separating gel was run with the sample loaded in a similar manner as described above. The precut blotting sandwich of the filter paper and PVDF was soaked in 100% methanol. The gel sandwich was prepared and then the gel and the filter paper were placed back to the cassette and the blot was run for 60 minutes at 100volts. Blot was stained with coomassie blue for 10 minutes, then rinsed with 50% methanol and then the blot was rinsed with 100% methanol. The membrane was placed at room temperature.

The blot was then rinsed with water, and the blot was blocked for 30 minutes with 10 mL, 5% blocking solution. The blot was then washed with 10ml PBST for 5 minutes. After washing, blot was placed in diluted primary antibody for 30 minutes. The blot was washed thrice with 10mL PBST for 5 minutes. After washing with PBST, it was placed in secondary diluted antibody and shake for 30 minutes. Again, the blot was washing thrice in 10 mL PBST and then finally, it was once washed with 10 mL PBST for 15 minutes. To develop western blot, solution I (9mL DI water, 1mL 1M Tris-HCL, 45μL Coumaric and 100 μL luminal) and solution II (9mL DI water, 1mL 1M Tris-HCL and 6 μL 30% H2O2) were mixed in a ratio 1:1. The blot was placed in this mixture for 1 minute. X-ray film was placed on the blot and the film was exposed in the developer solution until band appeared. The film was removed from the developer and rinsed with tap water. Then the film was placed in fixing solution until the film turn transparent, it was again rinsed with tap water and then rinsed with de-ionized water, and it was let dry.

Mass spectroscopy was performed to identify the exact molecular mass and formula of a specific protein. 10x Trypsin digesting buffer was used to digest the protein into smaller peptides. Samples were then loaded on the mass spectrometer following the similar procedure as described in Kevin, 2004.

Results:

Protein Concentration- (see appendix for sample calculations)

Table 1: Two method used, Bradford protein assay and Dumbroff/Gosh protein assay to determine the effect of different solvents on protein concentration.

Solvent Used

Bradford Method mg/mL

Dumbroff/Gosh Method mg/mL

100% Ethanol

0

0

Water

1.04

1.00

PBS

2.42

2.50

PBS +1% TritonX-100

3.61

4.00

PBS+ 2% SDS

4.98

5.00

The above table shows the effect of solvent on protein solubilizations using two different technique i.e Bradford protein assay and Dumbroff/Gosh protein assay. As indicated, PBS+2% SDS show the highest protein concentration using both methods. Dumbroff/Gosh method was proven to be a better protein quantification method as compared to Bradford protein assay method.

C:\Users\Kanwal Ash\Pictures\2011-01-31\001_crop.jpgFigure 1: Dumbroff/Ghosh blot to determine the protein concentration using different concentration standards and three different ratios of enzyme volume to 2% SDS volume. 1:1,1:10,1:150(mg/mL) respectively.

Western Blotting:

Results for α-GADPH Primary antibody

Western blot was performed in order to identify the protein after treating with α-GADPH antibody. The western blot image is shown below. The image indicates the appearance of bands for PBS+1% Triton X-100 and PBS+2% SDS at ~49kDa.

Lane

1 2 3

kDa

100

75

20

17

245

180

135

25

35

48

63

C:\Users\Kanwal Ash\Desktop\final pics\2011-March GADPH.jpg

Figure 2:Western Blot Gel after autoradiography: The image shows band positions of protein samples when treated with α-GDAPH antibody. Lanes from left to right represent: Lane 1 is Froggabbio protein molecular marker, lane 2 is PBS+1% Triton X-100 and lane 3 is PBS+2% SDS

Results for α-BSA Primary antibody

LaneWestern blot was performed in order to identify the protein after treating with α-BSA antibody. The western blot image is shown below. The image indicates the appearance of bands for PBS+1% Triton X-100 and PBS+2% SDS at ~66.2kDa. The results were consistent because theoretical molecular weight of BSA is ~66.2kDa.

1 2 3

kDa

25.0

35.0

45.0

66.2

116.0

Figure 3- 9% Western Blot Gel after autoradiography: The image shows band positions of protein samples when treated with α-BSA antibody. Lanes from left to right represent: Lane 1 is Fermentas SM 0431 protein molecular marker, lane 2 is PBS+1% Triton X-100 and lane 3 is PBS+2% SDS.

Results for α-β-actin Primary antibody

Western blot was performed in order to identify the protein after treating with α-β-actin antibody. The western blot image is shown below. The image indicates the appearance of bands for PBS+1% Triton X-100 and PBS+2% SDS at ~67kDa.

Lane

1 2 3

kDa

17

11

20

25

35

48

63

100

75

245

180

135

180 C:\Users\Kanwal Ash\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.Word\anti-BSA-afternoon.jpg

Figure 4- 9% Western Blot Gel after autoradiography: The image shows band positions of protein samples when treated with α-β-actin antibody. Lanes from left to right represent: Lane 1 is Froggbio protein molecular marker (4-20% Tris-Glycine), lane 2 is PBS+1% Triton X-100 and lane 3 is PBS+2% SDS.

Figure 5: The graph shows the relationship between the concentrations of the standards (mg/mL) with respect to corrected intensity using Dumbroff/Ghosh method.

Figure 6: The graph shows the relationship between concentration of different standards(mg/ml) and their absorbance in Bradford protein assay method.

Discussion:

According to the results shown in table I, Dumbroff/Gosh protein assay method showed better results i.e., high protein concentration for all solvents as compared with Bradford protein assay method. There is an apparent correlation between the type of solvent used and protein concentration. Studies indicate that water is a better solvent than 100% ethanol because it increases protein solubility 20 folds and present better results (6). The reason behind this is ethanol tends to lower the protein solubility because proteins after unfolding, re-fold themselves while exposing α-helices. Peptides are buried in the interior of these helixes, thus avoiding any contact with ethanol and gives poor protein solubility. As per results show, water produced 1.04mg/mL and 1.00mg/mL protein concentration for Bradford and Dumbroff/Gosh method respectively. Ethanol showed no protein concentration i.e 0mg/mL for both Bradford and Dumbroff/Gosh method. There was a gradual increase in the protein concentration as the percentage concentration of detergent increases. When PBS was used without any detergent, protein concentration for Bradford protein assay and Dumbroff/Gosh method was 2.42 mg/mL and 2.50mg/mL respectively. Although, there was not a significant difference between the two methods used but still Dumbroff/Gosh method better protein concentration. Also, there was not much protein concentration observed for both methods. The reason behind this could be that proteins are linked with many polypeptides chains and forms many covalent interaction i.e. disulfide bonds, which is harder for solvent such as PBS -a salt-water solution to break them at pH 7.4, thus produces poor protein solubiltity.

On the other hand, protein solubility increases as PBS along with other detergent i.e. 1% Triton X-100 and 2% SDS was used. When PBS+1%Triton X-100 was used, protein concentration for Bradford assay method was 3.61mg/mL and 4.00 mg/mL for Dumbroff/Gosh assay method. As said before, there was a gradual increase in the protein concentration when detergents were used. For instance, when compared there was an increase of 1.19 mg/mL protein concentration between PBS and PBS+1% Triton X-100 for Bradford assay method, and an increase of 1.50 mg/mL for Dumbroff/Gosh method. The reason behind this could be that Triton X-100 is a non-ionic detergent which readily solubilizes membrane proteins, thus tends to be a better solvent than PBS alone. A study shows that 0.015% of Triton X-100 produces optimum protein solubilization (17). Also, Triton X-100 does not ionize in aqueous solution because it does not affect disulfide bonds and hydrophobic interaction among peptides.

Among all the solvent used, PBS+2% SDS showed the highest protein concentration for either method. According to table I, protein concentration when PBS+2% SDS was used for Bradford assay was 4.98mg/mL and 5.00mg/ml for Dumbroff/Gosh assay. SDS is anionic detergent that is amphiphatic in nature and tries to denature the integral membrane protein. It makes bi-phasic interactions between protein-detergent. During its first phase, SDS tends to develop initial complexes which increase hydrophobicity, that results in turbidity and in the later phase it initiates solubilizing the protein, thus solubility increases. When compared, the results of protein concentration for both detergents along with PBS indicates that there was a difference of 1.37mg/mL between PBS+1% Triton X-100 and PBS+2% SDS for Bradford assay. Whereas there was difference of only 1mg/mL in protein concentration between the two detergents for Dumbroff/Gosh assays method.

Although the results showed better protein solubility for Dumbroff method but they were not that significant. This could be due to some experimental errors such as the amount of detergent used for solubilizing the protein would have not been enough. Another potential error could be that the presence of protease in the solution tends to degrade proteins therefore it is important to deactivate the protease quickly. Otherwise, protein degradation would result in lower solubility.

As expected, Dumbroff/Gosh protein assay proved to be a better protein quantification technique than Bradford protein assay. The reason behind this is that Dumbroff/Gosh method is not hampered by the presence of detergents as compared to Bradford assay. On the other hand, Ethanol did not produce any protein concentration, and the reason behind this could be that the timing was not sufficient for ethanol to solubilize protein. Also, there must be some contamination on Whitmann paper, or the amount of bovine liver homogenized was not just enough to produce satisfactory results.

SDS-PAGE technique was used to visualize the separated proteins corresponding to their electrophoretic mobility. As shown in image 1 (see appendix), the gel was stained with coomassie brilliant blue and silver stained. The result indicates that, coomassie blue gave more visible and separated bands than silver staining. Multiple bands were visible on the gel which indicates that several proteins were present in each solution.

As shown in the image 1(see appendix), there was a difference in band intensity between each solvent used. The highest band intensity and perhaps the darkest bands obtained were when PBS+2% SDS was used. The second highest solvent that produced darker bands was PBS+1% Triton X-100, then PBS and the lightest bands obtained were from water. This explains how changing the concentrations affect band intensity. Moreover, it was also observed that solvents, PBS, PBS+1%Triton X-100 and PBS+2% SDS almost travelled the similar distance as compare to water. The smaller the protein, the farthest it travels on the gel. According to this theory, PBS+2% SDS did not efficiently break the protein into its smaller peptides, due to experimental errors. Otherwise, ideally, peptides for this solvent would have travel the farthest on the gel and bands would have appeared at the bottom of the gel.

Unlikely, coomassie blue staining, silver staining did not show expected results and they are hard to interpret. The reason behind this is that silver staining is very sensitive and it even stains lower weight or smaller proteins which sometimes result in blurry or poor band visibility. However, according to the image 2 (see appendix) there was few observable trend of coomassie staining, i.e. there was a strong band visible at 66.2 kDa of the marker band. The band at 66.2 kDa of the marker SM0431 refers to BSA.

Figure 1-3 shows western blotting results. The protein markers used were Froggbio (pre-stained 4-20% Tris-glycine- see appendix) and Fermentas. Three primary antibodies were used α-GADPH, α-BSA and α-β-actin.

Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is a crucial enzyme in the process of glycolysis. This enzyme is able to bind with several proteins such as amyloid precursor protein(18). This is present at high concentration in cell and thus used as antibody. As shown in figure 2, there is a dark band corresponding to the molecular weight of ~49 kDa of a Froggbio marker. The expected result should be somewhere near 36kDa because that's GADPH molecular weight. The reason in this discrepancy could be that pre-stained markers often show some deviations (18).

Bovine serum albumin (BSA) is found in liver and it is used to distinguish between proteins because it is stable and does not affect different biochemical processes. The antibody used was α-BSA. As shown in figure 3, there was a strong band at 66.2kDa, which corresponds with the molecular weight of BSA. Presence of two solvents Triton X-100 and SDS affected the thickness and the number of bands appeared.

The results for α-β-actin antibody are shown in figure 3. For α-β-actin antibody, Froggabio marker was used. As shown in the image 4 (see appendix), dark band appear at ~67 kDa. The band should have corresponded to the β-lactoglobulin which is found in bovine milk. The results obtained were not consistent because α-β-actin has a molecular weight of 42kDa. The reason could be that sometimes bands that appear are slightly higher than their actual weight, due to the presence of chromophore in the pre-stained marker which causes deviations from actual results or there must have been some contamination.

Result of mass spectrometry is shown in image 4 (see appendix). It shows the identified protein with the detected mass to be 69278Da. The protein code was AAN17824 which represent serum albumin of Bos Taurus (see sppendix). Different proteins were also detected and are listed in appendix. The results obtained for mass spectrometry correlates with the outcomes of western blotting in previous findings. Therefore, the protein identified was BSA. Mass spectrometry is a better and faster technique than western blotting but it damages protein while sequencing.

Appendix:

Bradford Method:

Table 2: The table shows the data obtained to measure the percent transmittance at different standard concentration and the calculated absorbance.

Standard concentrations (mg/ml)

%Transmittance

Absorbance

0

100

0

0.01

93.5

0.030

0.05

79.5

0.100

0.10

71.5

0.145

0.50

39.0

0.410

1.0

19.0

0.721

1.5

10.0

1.000

2.0

6.0

1.221

2.5

4.0

1.400

5

2.8

1.553

Table 3: The table shows the data obtained to measure the percent transmittance using different enzyme to 1x PBS volume ratio and its calculated absorbance. The table also shows the concentration of the enzyme before/after the dilution with 1xPBS.

Ratio of enzyme to 1x PBS

%Transmittance

Absorbance

Concentration after dilution (mg/ml)

Concentration of original sample (mg/ml)

1:1

60

0.222

0.393

0.786

1:10

85

0.071

0.126

1.26

1:150

89

0.051

0.090

13.5

Sample Calculation for 1:1 Ratio:

Dumbroff/Gosh Method

Table 4: The table shows the data obtained from calculating the area of the spot, mean, absolute, relative and corrected intensities based on the concentrations of protein samples given using Dumbroff/Ghosh method.

Conc. Of different sample (mg/mL)

Area of the spot

Mean

Absolute Intensity

Relative Intensity

Corrected Intensity

0

4203

5.897

24785

1

0

0.01

6009

9.970

59910

2.417

1.417

0.05

6660

16.215

107995

4.357

3.357

0.1

4190

21.577

90409

3.648

2.648

0.5

3783

60.093

227332

9.172

8.172

1.0

4068

89.884

365649

14.753

13.753

1.5

4466

114.793

512665

20.685

19.684

2.0

4203

125.005

525397

21.200

20.198

2.5

4422

132.225

584701

23.591

22.590

5

3809

147.395

561428

22.651

21.651

Table 5: The table shows the data obtained from calculating the area of the spot, mean, absolute, relative and corrected intensities based on the concentrations of proteins before and after the dilutions.

Ratio of enzyme to 2% SDS

Area of spot

Mean

Absolute Intensity

Relative Intensity

Corrected Intensity

Concentration after dilution (mg/ml)

Concentration of original sample (mg/ml)

1:1

1392

109.470

152382

6.148

5.148

0.490

0.980

1:10

4443

43.278

192282

7.758

6.758

0.643

6.430

1:150

3665

8.843

32408

1.310

0.318

0.030

4.388

Sample Calculation of sample 1:1 Ratio:

Lanes

1 2 3 4 5

kDa

45

116

66.2

35

25C:\Users\Kanwal Ash\Desktop\final pics\PM -- Kanwall + Mamoona.tiff

Image : 9% SDS-PAGE stained with Coomassie brilliant blue: The image shows the bands appearance for different solvent.Lanes from left to right represent: Lane 1 is molecular marker Fermentas SM 0431, lane 2 is water, lane 3 is PBS+1% Triton X-100 and lane 4 is PBS+2% SDS.

Lanes

1 2 3 4 5

18.4

kDa

45

116

66.2

25

35 C:\Users\Kanwal Ash\Downloads\fwchy362_w11_01silverstaininggelscans_(1)\Andy+cody.tif

Image 2: 9% SDS-PAGE stained with silver staining technique: The image shows the bands appearance for different solvent.Lanes from left to right represent: Lane 1 is molecular marker Fermentas SM 0431, lane 2 is water, lane 3 is PBS+1% Triton X-100 and lane 4 is PBS+2% SDS. **(image taken from Jaspreet section).

Image 3: The image shows the peptide report from Mass spectrometry. The protein code is AAN17824- Bos Taurus.

PROTEIN MARKERS:

SM0431 (19)

Froggabio (20)

C:\Users\Kanwal Ash\Desktop\final pics\391.jpg

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