Introduction:
Western blot is an analytical technique used to detect specific proteins in a given sample. It consists of diverse applications to investigate certain adaptations, for example, protein-protein interactions and protein abundance. Western blot experiments involve direct versus indirect detection. When using the “direct detection method, an enzyme- or fluorophore-conjugated primary antibody is used to detect the antigen of interest on the blot” (Overview). The direct method is not preferred by researchers for various reasons. In indirect detection an unknown antibody is first used to bind to the antigen. Indirect method has many more advantages, such as signal amplification from the secondary antibody, a single secondary antibody can be used with a number of primary antibodies and using secondary antibodies does not inhibit primary antibody target binding. The direct method is unlikely be used due to being costly, limited, and potentially interfere with target binding.
Antibodies are blood proteins produces in response to counteract a specific antigen. Antibodies can also be known as immunoglobins which are produced by the immune system to help stop intruders from harming the body. These intruders can be viruses, bacteria, or other chemicals. When an antigen is found in the body, the immune system creates antibodies to “mark” the antigen for the body to destroy it. Each antibody is made for only one antigen, and it is fitted with special receptors with will bind to only that antigen. For example, a specific antibody is created to help destroy measles/mumps/rubella- commonly known as MMR. Only that particular antibody will attack MMR. Once the antigen tries to enter the body the immune system gets triggered. Chemical signals alert different parts of the immune system into action. The virus is met by B cells, which are responsible for creating antibodies to match the antigen. After the antibodies are created and stick to the virus- T cells then attack the antigen. Phagocytes, or large cells come to eat remains of the infection once the antigen has been destroyed. There are five types of antibodies, Iga, IgD, IgE, IgG, and IgM. Each of the immunoglobins are located around different parts of the body with different duties from one another.
A human’s immune system is fully developed by six to nine months, this is where children response to more strongly glycoproteins. Not until a child of 1-2 years of age is marked as improvement in body response to polysaccharides. Polysaccharides are carbohydrates molecules that have sugar molecules bonded to it. CDC recommends vaccinations before the age of two years to protect children against infectious diseases like varicella, polio, influenza, and twelves others. When a child is born, doctors given the newborn their first dose of Hepatitis B. At two months, the baby has had their second dose of Hepatitis B, Diphtheria, tetanus, and acellular pertussis vaccine (DTaP), Haemophilus influenzae type b vaccine (Hib), Inactivated poliovirus vaccine (IPV), Pneumococcal conjugate vaccine (PCV), and Rotavirus vaccine (RV). As you can see a child at such a young age undergoes lots of immunizations to boost their immune system. Meanwhile, a cow develops antibodies immediately after it is born Actually calves produce antibodies in the uterus because it is exposed to pathogens. Which means that a calf has a substantial amount of protective immunity by its fourth week of life. A clean and hygienic place is very important for the newborn for any animal because of the pathogens located in a dirty environment can be toxic to babies.
Serum from fetal, newborn, and adults’ cows are applied to undergo nondenaturing gel electrophoresis conditions. After electrophoresis, the gel is then pressed against nitrocellulose paper. This is where proteins from the gel stick to the nitrocellulose. Next, the nitrocellulose is incubated with excess of gelatin, which blocks nonspecifically to protein binding sites on the nitrocellulose. The nitrocellulose is later incubated with rabbit antibodies against cow immunoglobulin. The rabbit antibodies have previously been conjugated with peroxidase. When peroxidase substrate- a colorless solution is added, the appearance of colored product identifies the cow immunoglobulins that have been bounced to antibodies.
Methods:
To obtain the results in Figure 1, samples were put into a 0.8% agarose gel and run at 170V for about 20 minutes. Once this was complete a “sandwich” was created with one sheet saturated with TBS agarose gel face down, nitrocellulose paper wet from water and TBS, another sheet of TBS blotting paper, two sheets of dry blotting paper, and lastly 1 cm thick of stacked paper towels. Pressure was exerted on this “sandwich” for 3 minutes. Once complete the “sandwich” was disassembled and the nitrocellulose paper was stained in blot stain for 5 minutes, then rinsed 3 times. This nitrocellulose can be seen in Figure 1.
To obtain these results in Figure 2, nonspecific protein binding sites were blocked by incubating the nitrocellulose from Figure 1 in gelatin-TBS for 10 minutes at 37℃, then with fresh gelatin-TBS for another 10 minutes. The nitrocellulose was incubated later in rabbit antibodies against cow immunoglobulin conjugated with peroxidase for 20 minutes at 37°C. A series of 3-minute washes with continuous rocking of TBS/NP40 three times ensued, along with one wash of TBS. Then the nitrocellulose was incubated for 5-15 minutes in a peroxidase substrate (colorless) causing a purple color to form. The nitrocellulose was rinsed three times with water. This process then identified the cow immunoglobulins that have bound antibody to immunoglobulin, as shown in Figure 2.
Results:
Figure 1. Nitrocellulose Paper Protein Stain (10/9/18)
Proteins from the samples run in the gel stuck nonspecifically to the nitrocellulose paper. Lanes 4, 6, 8, and 10 were left blank. Cow transferrin (T) (lane 1) is a protein that binds iron, but none of this protein stuck to the nitrocellulose paper. Cow albumin (A) (lane 2) is a major protein of serum. No cow albumin protein was present on the nitrocellulose paper. Cow gamma globulins (G) (land 3) are antibodies and immunoglobulin. There was some G protein present after gel electrophoresis on the nitrocellulose paper. Fetal calf serum (FCS) (lane 5) protein was present on the nitrocellulose. Newborn calf serum (NCS) (Lane 7) protein was not present on the nitrocellulose. Adult cow serum (S) (lane 9) protein showed on the nitrocellulose in a large amount. These results indicated that these samples had proteins present within them by nonspecifically binding to the nitrocellulose indicating an affinity. This technique also ensures that the protein, immunoglobulin, is present on the nitrocellulose for future steps when it is trying to be identified within the samples.
Figure 2. Nitrocellulose Paper Cow Immunoglobulin Stain (10/16/18)
Nitrocellulose was incubated in gelatin to block nonspecific protein binding and incubated in rabbit antibodies conjugated to peroxidase against cow immunoglobulin. A purple product could be seen after the addition of the colorless peroxidase substrate, identifying cow immunoglobulins that have bound antibody to immunoglobulin. It was shown that adult cow serum had the most immunoglobulin present as it had the darkest and largest purple band indicating the most antibodies present as well (Lane 9). Newborn calf serum had immunoglobulin and thus antibodies present, but it is not as pronounced (Lane7). Fetal calf serum had even less immunoglobulin and thus less antibodies present (Lane 5). Cow gamma globulins had immunoglobulin and antibodies present as well (Lane 3). Cow transferrin (Lane 1) and cow albumin (Lane 2) showed no immunoglobulins present indicating no antibodies present. Lanes 4, 6, 8, and 10 were left blank. These results insinuate that overtime during calf development, immunoglobulins and antibodies continue to develop and increase as well.
Conclusion:
These results indicated that these samples had proteins present within them by nonspecifically binding to the nitrocellulose indicating an affinity. This technique also ensures that the protein, immunoglobulin, is present on the nitrocellulose for future steps when it is trying to be identified within the samples.
Successfully after the nitrocellulose was incubated in gelatin to block nonspecific protein binding and incubated in rabbit antibodies conjugated to peroxidase against cow immunoglobulin. A purple product received after the addition of the colorless peroxidase substrate, identifies cow immunoglobulins that have bound antibody to immunoglobulin. As shown in Figure 2 the adult cow serum had the most immunoglobulin present as it had the darkest and largest purple band indicating the most antibodies present. The newborn calf serum had antibodies present, but it is not as likely. The fetal calf serum had even less immunoglobulin and thus less antibodies present. Cow gamma globulins had immunoglobulin and antibodies present. Cow transferrin and cow albumin showed no signs of immunoglobulins present, this indicating no antibodies present.
Discussion:
Gel electrophoresis is common laboratory method used to separate mixtures of DNA, RNA, or proteins. When the gel runs for about 20-30 minutes, molecules separated are pushed by electrical through the gel that contains small pores.
Nitrocellulose could not be handled with bare hands because it is a highly flammable material made by treating cellulose with concentrated nitric acid. This is the reason why the lab manual indicated to wear gloves or use forceps. The nitrocellulose is fragile and somewhat brittle, but very powerful and dangerous because it can be used to make explosives and celluloid.
This experiment was used to identify cow serum immunoglobins using western blot. As mentioned previously antibodies are blood proteins produces in response to counteract a specific antigen, to stop intruders from harming the body. A human’s immune system is fully developed by six to nine months, this is where children response to more strongly glycoproteins. When in comparison a cow’s immune systems starts to activate in the uterus. Results were received successfully as shown in Figures 1 and 2. The samples given to us had proteins present within them by nonspecifically binding to the nitrocellulose indicating an affinity. When the peroxidase substrate was added, a colored product appeared, which identified the cow’s immunoglobulins that have bounded antibodies to immunoglobulin.
“An individual can have components of immune function that they produced themselves, also known as active immunity or immune components produced by another individual- also known as bassive immunity. In many mammals, active production of antibodies first begins during the months after birth. Thus, passive immunity provides protection in early life. There are two natural sources of passive immunity in mammals. In some, but not all, species matemal antibodies are transferred from mother to fetus across the placenta. Also, antibodies are transferred from mother to newbom through the milk” (Lab Handout).
If improvements had to be done, collecting our own samples could be helpful for better results in case of any human error occurred. During the “Blotting Proteins onto Nitrocellulose” section could have been done better for clearer results. Personally, I think the weight on the “sandwich” could have been a bit heavier for more pressure exerted so that proteins stick nonspecifically better on the nitrocellulose paper. The color development was very successful due to the quick color development.
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Bibliography:
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2. Driessen, J. (n.d.). At which age do calves starts with antibodies production in its immune system? / CowSignals Training Company. Retrieved from https://www.cowsignals.com/blog/at_which_age_do_calves_starts_with_antibodies_production_in_its
3. Overview of Western Blotting. (n.d.). Retrieved from https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html
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