Proteomics Lab Report
Proteomics Lab Report
Proteomics Lab Report
2092434
iii. SDS-PAGE
SDS-PAGE is the second dimension for protein seperation. It is so far the most employed technique for
quantitative expression profiling of protein mixtures including cell lysates and it allows for the seperation of
proteins based on their pI, molecular mass, solubility etc. Gel has been prepared by bisacrylamide and
acrylamide and polymerased with TEMED which is the polymerizing agent. Proteins are denatured and by
the addition of sodium dodecyl sulfate and mixed with loading solution that has bromophenol blue dye. It
acquire a net negative charge proportional to their length. The gel has been subjected to an electric field
which allows to the migration of proteins. Smaller proteins are migrated and moved further on gel. The strip
that is obtained before, putted on top of the SDS-PAGE with the use of agarose and bromophenol mix,
stabilized. And the system connected to the power source and runned.
3. Further Analyses
a. Gel Staining
In order to prevent protein diffusion, the proteins that are seperated acquires fixation. We
achieved fixation by ethanol and acetic acid. Those fixatives helps to maintain the
position of the proteins and prevents passive diffusion overtime. Protein visualization is
performed by Coomassie staining and Silver staining. Coomassie dye binds to the
proteins that are fixed and forms blue color and in order to remove excess dye on gel
double distilled water and destaining solution has been used. At the end, the intesity of
the color gives information about relative abundance but since we need more sensitive
techniques, we performed silver staining. It is a sensitive technique for protein
visualization. We placed the gel into silver nitrate solution. By that, silver ions binds to
amino side chains, particularly sulfhydryl and carboxyl groups of proteins. Aterwards,
we used developing solution which contains formaldehyde and sodium carbonate and by
that the silver-protein complex that we obtained as a result of the binding of silver ions to
proteins are reduced to metalic silver and the reduction occurs at the sites of protein spots
on the gel and it leads to visible silver deposits that makes protein spots/bands visible. At
the end, in order to stop the reaction, we used stopping solution which includes acetic
acid. By that, we prevent further silve accumulation and stabilized already developed
bands. Then we used fixing solution to preserve the integritiy of the bands and diffusion.
b. Immunobased Assays
i. Western Blotting
Western blotting is also known as immunoblotting and it is commonly used to
detect specific proteins in a mixture of proteins. The main principle behind is
antigen-antibody interactions. After the protein seperation has been done, we used
the gel that we obtained through electrophoresis are hydrated through water to
prevent drying. In order to transfer proteins on the gel to a nitrocellulose paper we
prepared a sandwich which consists of nitrocellulose paper, sponge, absorbant
paper. After we prepared the sandwich and clipped all together and placed it in
electrophoresis chamber.Then we added running buffer to chamber and ice to
prevent over heating and run it. After the transfer has been achieved, the membrane
is then incubated with labels antibodies specific to the protein of interest. The
unbound antibody is washed off leaving only the bound antibody to the protein of
interest.The bound antibodies are then detected by developing the film (Mahmood T
et.al, 2012). In order to visualize proteins, reversible Panceau S staining has been
used.
4. Biomarker Identification
In proteomics, protein discovery and characterization rely on mass spectrometry (MS) techniques. In a
suitable procedure, proteins are separated and analyzed by MS. These data are compared with sequence data
using Mascot and other search engines to identify proteins. In peptide mass fingerprinting (PMF), unknown
proteins are extracted and digested into peptides and analyzed by MS to identify proteins. Peptide
fragmentation fingerprinting (PFF) Tandem MS (MS/MS) for fragment ion data detects the protein . In both
methods, the protein sequence must be present in the database. Post-translational modifications (PTMs) are
critical for protein function. MS characterizes PTMs based on significant changes in modified amino acids.
However, PTMs are often present in such low abundance that amplification methods are used. MS ionized
analytes and seperates them based on their mass to charge ratio using mass analyzers. The ion current is
detected and producing a mass spectrum (J. Gallardo, et al, 2013).
5. Results and Discussion
6. References
Gallardo, J. M., Ortea, I., & Carrera, M. (2013). Proteomics and its applications for food authentication and food-technology research. TrAC - Trends
in Analytical Chemistry, 52(December), 135–141.
Gašo-Sokač D, Kovač S, Josić D. Application of Proteomics in Food Technology and Food Biotechnology: Process Development, Quality Control and
Product Safety. Food Technology & Biotechnology [Internet]. 2010 Jul [cited 2023 Jul 3];48(3):284–95.
Giacometti, Jasminka, Alena Buretić Tomljanović, and Djuro Josić. "Application of proteomics and metabolomics for investigation of food
toxins." Food research international 54.1 (2013): 1042-1051.
Görg, A., Weiss, W., Dunn, M. J. (2004). Current Two-dimensional Electrophoresis Technology For Proteomics. Proteomics, 12(4), 3665-3685.
https://doi.org/10.1002/pmic.200401031
JoVE Science Education Database. <em>Biochimica.</em> Photometric Protein Determination. JoVE, Cambridge, MA, (2023).
Mahmood, T., Yang, P. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North Am J Med Sci, 9(4), 429.
https://doi.org/10.4103/1947-2714.100998
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