Lab report discussion and results
Introduction
The experiment is aimed at studying about the protein content in the cells and tissues of various organisms and the ways of regulating the growth of some tissues in the body of the animals like in our case we used the rabbit tissues in determining the feasibility of the experiment. The process begins with the ability to look at the process of obtaining the protein from the sample given and how to quantify the amount of protein in such cells.
Method:
Protein extraction
The process of protein extraction involves three major processes which must be followed in order to accomplish the process of protein extraction needed to carry out any study about protein. These are:
- Collection of the tissues or cells rich in protein
- Lysis of the cells.
- Separation of non-soluble components especially the non-protein parts; through centrifugation process.
The experiment was aimed at attaining the results in checking for the amount of the protein content in any tissue or cells of any animal like rabbit for instance. It is by use of the simple means of western blotting. This is the most important technique normally used in the detection of the protein in a mixture of a given sample. The technique can be able to allow the quantification of the protein content in the cell or tissues. It is believed that protein quantification is one of the vital aspects involved in the protein content determination in any sample. This is majorly used in the protein extraction, purification and even the issue of protein sampling. Western blot is at times referred to as the immunodetection technique used to detect and quantify specific proteins in biological samples.
Western blotting normally bases its method of cases like the molecular mass and the issues of antibody binding specificity ability. These makes the activities of the particular protein be determined by the researcher so easily since he or she can be able to monitor the movement of the blot and come up with a deduction. For better results, we discovered that blot must be incubated in a protein rich solution to block all necessary non-specific binding sites. Every type of protein assay is adversely affected by substances of one sort or another.
The components of any protein sample solution was considered interfering substances in a protein assay if they artificially suppressed the response, enhancing the response, or caused elevated background by an arbitrarily chosen degree. From experiment carried out, we learnt that it is vital to western blot procedure to perform a separation of macromolecules first before the actual determination of the protein quantity in any sample. This can be done by use of the gel electrophoresis process, in which after the process the separated molecules are transferred into a blotted onto a second bit of the matrix of the membrane and it is blocked from any non-specific binding after that separation.
Literally, western blotting is believed to be a six- stepped technique which was able to take the desired protein and then transfer it from the gel given then finally to the marked antibodies field. This helped in determining the various similarities the proteins do have in common depending on their lengthy aspects that they have. From our study it was vital to come up with a conclusion that western blotting varies with respect to the type of the medium in which the sample is put in determining the protein quantity and may bring different but almost closely related results to the researcher.
For the control experiment in better determination of the precise results of the experiment, a control experiment was laid down and their results were arrived at as per the figure below:
Western blot analysis of 10 uL T74D using p44/42 ERK Rabbit mAb. HCT (+) FBS, HCT (-) FBS were used as controls.
For example the graph shows the results obtained on determining the effects of protein concentration on the absorbance in Bradford protein Assay. Known concentration of bovine serum albumin (BSA) was used to construct a standard curve for calculation protein concentration of cell extracts. This gave varied results when plotted to obtain a graph and showed that it is directly proportional to the protein concentration since it almost gave a straight line though not via the origin.
Effect of proteins concentration on absorbance in Bradford protein Assay. Known concentration of bovine serum albumin (BSA) was used to construct a standard curve for calculation protein concentration of cell extracts.
Result of the protein extraction:
The concentration of the unknown protein is 0.391 ug/ul , while the total yield is 97.85 ug. From the result shown by the table, it is clear that the concentration is directly proportional to the amount of the cell extracts, though it does not pass through the origin of the graph.
The use of the SDS-PAGE in the experiment was vital since it helped to bind the protein with the negative charges. It is an ionic detergent and so helps in the denaturing of the proteins and coats the content with the negative charges preventing further unnecessary binding by the unspecific substances. This still helps in the separation since most of the required substances will be migrated towards the positive sides of the experiment enhancing the separation of the content needed.
In concluding, it was vital to note that western blot is a technique normally used in protein analysis and detection as it gives the user to get the exact quantity of the protein expression in the sample in question. The experiment made the research about the protocol, the theory behind that protocol, and some very important issues of concerns in the techniques. It could have been as an intricate measure, as we made attempts to get a nonspecific binding factor, yet stronger signal in return.
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