Abstract

Protein separation and identification is an important process in both medical and veterinary fields. Precision and accuracy are important so that the correct identification could be made. Electrophoresis and western blotting are efficient ways to carry out the process. In this experiment, they were used to analyse a sample in order to determine the presence of protein. Electrophoresis uses charges for separation. At the end of the experiment, it was determined that the sample under investigation contained protein chicken IgG. The protein was further examined to determine the light and heavy chains of polypeptides L and H. The method was, therefore, effective due to its quick and accurate results. Examples of its application in both human and animal medical fields were then analysed. Its advantages over other methods as well as its disadvantages were highlighted. This ensured a deep understanding of the process with its pros and cons for using it in perspective. Finally, a conclusion was drawn based on experiment findings, discussion, and the existing knowledge.

Introduction

Western blot, also known as protein immunoblot, is an analytical technique used to detect specific proteins in a sample of tissue extract or homogenate. It uses gel electrophoresis to separate native proteins using 3-D structure or denatured proteins following the polypeptide length (Harlow & Lane 1999). A transfer of proteins to a membrane is then done. After that, proteins are stained using antibodies that are known to be specific to the particular protein. This method is very effective and the most accurate of the available protein detection methods, especially in small amounts. The process is also able to determine other features such as the half-life of the proteins as well as molar amounts. The use of ELISA ensures higher specificity. Therefore, the method is more independent to the antibodies (Towbin, Staehelin, & Gordon 1979; Harlow & Lane 1999).

The method can also be used to isolate immunogenic responses from infectious agents with the exclusion of viruses and bacteria. These are hard to isolate from a sample using other methods. Nevertheless, western blotting can effectively carry out the process.

The method also uses antisera as its diagnostic tool. This is in addition to using antigens, which other methods use. In this experiment the process was more accurate than any other and this resulted into more accurate results and conclusions.

This experiment was carried out in two phases: characterisation of immunoglobulins and polyacrylamide gel electrophoresis. Immunoglobulins (Ig) are multi-chain glycoproteins. They are covalently bonded (disulphide bond) with an association between two identical heavy and two light polypeptide chains, denoted as H and L respectively. When used in SDS polyacrylamide gel and non-reducing conditions, Igs moves according to molecule sizes. After electrophoresis, however, the molecule dissociates to its constituent heavy and light chains (Renart, Reiser, & Stark 1979).

Electrophoresis is used to separate charged molecules according to their migration ability when placed within an electric field. This process usually uses polyacrylamide gel electrophoresis (PAGE), where proteins are moved through a cross linked by a gel, organic molecule, which sieves protein molecules, allowing some and disallowing some through it.

Protein separation uses alkaline buffers to ensure that proteins get negatively charged, thus move to the positive end. When sodium dodecyl sulfate (SDS), a detergent that is usually negatively charged, is used, proteins can be separated according to their molecular masses. SDS-PAGE, uses molecular shapes of proteins as a base for separation. SDS molecules that bind to a protein usually match protein’s molecular mass. Eventually, the size does not affect proteins’ movement since they all have a similar charge. Smaller proteins move faster due to the gaps between ‘sieves structure’ (Kamps & Sefton 1988). It is, therefore, effective to separate proteins using their molecular masses. During this experiment, a polyacrylamide gel was prepared and gel electrophoresis used to identify the details of immunoglobulin identity and structure. The specific molecule was then determined.

Immunoblotting techniques perform the process of protein separation, which is later followed by immunodetection. Polyacrylamide gel is used in western blotting to separate the substance into component proteins, which are then transferred to a blotting medium (a nitrocellulose or PVDF membrane). The blot is then subjected to an antibody that works with an antigen in the substance under investigation.

Immunoblotting is often used as a stand-alone spot test due to its high precision and reliability (Harlow & Lane 1999). This study used western blotting to confirm the identity of proteins that were analysed by gel electrophoresis in the first stage of this experiment.

Materials and Methods

Phase I Characterisation of Immunoglobulins

The following requirements were assembled before the experiment: polyacrylamide gel solution (Next Gel 12.5%; Astral Scientific), 3 gels per 20 ml, ammonium persulphate and TEMED tablets (Astral Scientific) (add 1 tablet to 1ml water and dissolve before use – students to prepare; only require 2 separate tubes), immunoglobulin samples (bovingIgM, bovine IgG, chicken IgG each @ 1mg/ml), molecular weight marker (kaleidoscope; BioRad) + specification sheet with known molecular weights, electrophoresis running buffer (Next Gel Running Buffer, 20X, Astral Scientific), loading buffer – reducing (62.5mM Tris-HCl pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 0.2M dithiothreitol; DTT), biosafe protein stain + stain/destain trays, gloves, heating block, microfuge, ice/cooling blocks for 1.5ml tubes, 1.5 ml sterile centrifuge tubes, transfer pipettes, gel-loading tips and/or yellow tips for loading gels, micropipettes, yellow pipette tips, gel casting and electrophoresis equipment (Biorad casting stands, vertical electrophoresis tanks and power packs), and white light box.

Safety measures for the experiment were thoroughly adhered to, especially before starting to handle acrylamide solutions and gels and the biological detergent - SDS. After all requirements to run an electrophoresis experiment were assembled together, groups of four students were created. Every student was expected to load at least one sample of gel. Gel casting apparatus was then constructed. The casting stand was held together using a clamp.

200μL of APS/TEMED was added to 20 ml of Next Gel solution. Thorough mixing using a transfer pipette was done. Mixing was done carefully to avoid air bubbles.

By using a transfer pipette, the space between glass plates was filled with the acrylamide solution to the top of glass plates (making sure to avoid air bubbles) and the comb was carefully inserted between glass plates (the comb formed the wells used to load protein samples into). The comb was then left in place for 45 minutes for the gel setting.

To prevent air bubbles from interfering with gel setting, the gel was overlaid with a prepared agar solution with 3 gels being obtained from every 20 ml of the solution. The rest of the gel was left to be later used to determine the set rate of gel. As the gel settled, the running buffer was made.

Once gels were set, they were put into the gel holder, combs were removed, and gels placed in the tank. The buffer was added to the ‘internal tank’ and used to rinse the wells to clear them from excess acrylamide solution. Once all wells were covered, buffer was added to the external tank to cover electrodes by 2cm.

30μL samples (chicken Ig, bovine IgG, bovine IgM) for loading on gel were then prepared by diluting 10μL of each Ig sample 1:2 in loading buffer (10μL + 20μL). All samples were heated in a heating block at 95°C for 5 minutes. The tubes were secured by using a cap closer. Samples were then cooled on ice and quickly centrifuged to collect contents to the bottom of the tube. They then were stored on ice until the sample was loaded on gel.

20μL of treated sample was then carefully loaded in the wells alongside 10μL of molecular weight marker per group. The gel tank was then set up, checked by the tutor, and the power pack turned on.

The gel tank was set up and electrophoresis was done at 150V for 55 minutes until the dye front reached the bottom of the gel. After that, the power was turned off and gel was removed from the gel apparatus, leaving the gel ready to use.

Phase 2 and Polyacrylamide Gel Electrophoresis

These requirements were assembled for the electrophoresis phase of the experiment: western blotting gel tank and accessories, blotting membrane (PVDF) and filter papers cut to size, transfer buffer (Next Gel), washing buffer (0.05% Tween 20 in 1x PBS), shallow trays (large enough to hold and wash the blot), blocking solution (5% skim milk powder in washing buffer), top-pan balance, gloves, orbital shaker, magnetic stirrer with very small stirrer bar, cold blocks, horseradish peroxidase conjugated probing antibody (anti-chicken IgG at 1:2000 in blocking solution), substrate (DAB), tweezers for blot handling, and a piece of foil.

The experiment was done carefully avoiding contact of the substance with the skin. Protein gel prepared in Experiment 4 was removed from between glass plates and the wells were neatly trimmed. The gel was then immersed in transfer buffer for 30 minutes. Filter papers were then soaked in transfer buffer for 30 seconds. The membrane was out in methanol for 30 seconds and then rinsed in milliQ water for 2minutes.

The membrane was placed in transfer buffer and equilibrated for 5 minutes. The stack was then assembled as follows with the membrane placed on the anodic side of the tank. The arrangement from the negative to positive sides was, therefore, as follows:

Direction of transfer ↓↓

(-) Cathode (black)

Foam pad

Filter paper

Gel

Membrane

Filter paper

Foam pad

(+) Anode (red)

The cassette holder was opened and foam fibre pad was placed on one side of the cassette. In steps that followed, each of the layers was smoothened to avoid air bubbles on the blot.

A sheet of filter paper was placed on top of the pad, gel was placed on top of the filter paper, and the membrane was placed on top of gel. A second sheet of filter paper was then placed on top of the stack and a second foam fibre pad was placed on top of the filter paper. The cassette holder was closed and placed in the transfer tank ensuring that the gel side of the cassette holder was facing the cathode (-) and the membrane side was facing the anode (+).

Transfer buffer was then added to the tank to cover the cassette holder, and then a stirrer bar was added to the tank. The tank was then placed on the stirrer so that heat was transferred through the tank during the run.

The leads into electrode jacks were inserted on the transfer unit and connected to the power pack, then 18. Run for 20 minutes at 2.50AMP. Once the run was completed, the apparatus was disassembled. The blot was always handled using forceps. Immunodetection phase was carried out on an orbital shaker at room temperature. Careful attention was paid to ensure that the bolt was never allowed to get dry during the experiment. The blocking solution was prepared 30 minutes before it was used.

Membrane was placed in trays with the blot side up and then covered using 50mL blocking solution. The set up was then agitated on an orbital shaker for 60 minutes at room temperature, ensuring that the blot was covered in solution at all times.

The blocking solution was removed and washed with washing buffer for 10 minutes on the orbital shaker. The wash was then discarded and the blot overlaid with 50 ml conjugated primary antibody (HRP conjugated anti-IgG 1:2000 in blocking solution) solution and incubated for an hour on orbital shaker.

The blots were then washed for 20 minutes in the washing buffer. 30 ml of DAB substrate was then prepared as instructed. The blot was placed in a clean tray and completely covered with the substrate solution. It was then covered with foil until bands were visible on the blot for 4 minutes. The set up was then washed in distilled water several times and positions of bands relative to the molecular weight marker were noted. The position of  bands on the blot was compared to the stained protein gel and protein samples were identified in the original tubes.

Results

The western blot experiment resulted in two columns. The first column had several stains that basically indicated the presence of protein in the sample. It had pink stains due to the kind of buffer used to mark the presence of the chicken IgG protein. The second column from the blot experiment had two unclear stains that identified the presence of other substances apart from protein. This was because the process did not dissociate components of protein. However, in the second phase of the experiment, other columns and dots were observed on the blotting paper. They were able to determine other substances available such as 50Kda and 25Kda, which were heavy and light chains in the sample respectively. They were a breakdown of chain components of protein.

The dyes were used to track and monitor the movement of substances in the sample. By the time the dye front got to 80%, the process could be stopped since substances would have, in most cases, differentiated. This, however, is dependent on the sample molecular weight. During the experiment, the SDS was used to bind to protein to make it anionic. This ensured that the sample moved with respect to its molecular weight. Stains on the blotting paper showed the presence of chicken IgG. The experiment confirmed the presence of chicken IgG, which constituted of heavy and a light chain.

Discussion

The used method was effective in determining the presence of proteins in the sample provided. There were very clear marks on the scale that showed the presence of three substances in the sample. Chicken IgG dominated the constituents. There were no uneven marks a well as patches due to high precision and efficiency of the method. This method can be effectively used to determine the presence of little quantities of proteins in any sample.

The method can be used to detect anti-HIV antibody found in humans using samples of serum. Serums are tested to determine which contains the antibody. It can also be used as a definitive test for the mad-cow disease, Bovine spongiform encephalopathy, BSE as well as Lyme diseases. It has also been used to confirm the presence of FIV+ status in cats (Bolt & Mahoney 1997).

Advantages of this method can be explained by its popularity in confirmation tests as indicated above. It is accurate and effective and goes deeper into detecting other features of proteins such as half-life and other infectious agents via their immunogenic responses (Hames & Rickwood 1998). Further, it uses antisera as a diagnostic tool and not antigens alone. It also uses cheap materials such as polyvinylidene difluoride (PVDF) or nylon as membrane due to its high ability to bind protein and its chemical stability. It is also quick despite its high precision (Bolt & Mahoney 1997).

Apart from advantages, the method has also several disadvantages. The latter show where protein not intended for the experiment may be detected due to its high sensitivity. This may lead to labeling of the wrong protein (Hames & Rickwood 1998). At the same time, oxidation of proteins could result in emergence of multiple bands, thus the precision of the method is lost. Further, bubbles that have to be avoided during the transfer of the sample from the gel may result into poor results. Timing is also vital and, if planned poorly, may lead to inaccurate results.

Conclusion

Western blotting is a very effective method for determining the presence of proteins in a sample. It is accurate and reliable and can be used at any level of medical practice. However, it requires a lot of precision in processes such as correct measurements of required substances. Furthermore, air bubbles during the transfer of the sample to the gel will result to inaccurate and unreliable results. It is, therefore, a very delicate process that can be nullified by simple mistakes. To ensure that the best results are obtained from the method, one needs to perform the experiment several times to reduce the degree of error. Despite the sensitivity of the process, it still remains among the most credible methods to determine the presence of proteins, especially in samples that are believed to have very little amounts.

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