Difference between revisions of "Team:NAIT Edmonton/Desc"

The structural and functional study of the proteins expressed by a genome is called proteomics. This relatively novel science uses different methodologies in order to separate and identify specific proteins of interest. Among these techniques, SDS-PAGE plays an essential role due to its high sensitivity, low sample volume requirement, and high popularity. Negatively charged proteins migrate towards the positive electrode according to their size and charge. Smaller proteins migrate further in a given amount of time. As proteins are separated in this manner, users load molecular weight standards to estimate the size (in kDa) of the proteins present in their sample. Once the proteins of a single sample have been isolated and are embedded in the polyacrylamide (PA) gel matrix, staining procedures are used to visualize them.

Source: http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png

Organic dyes, such as Coomassie blue, can be used for this purpose; nevertheless, their low sensitivity and a detection range that goes from 1 to 50 ng can be a challenge for detecting low abundance proteins (Jin, Huang, Yoo, & Choi, 2006). A higher sensitivity can be achieved by fluorescent staining techniques (from 0.1 to 10 ng.); however, UV instruments are necessary in order to read the data (Jin et al., 2006). The most sensitive method up to date is radiolabeling, but the requirement of hazardous isotopes and their complex management makes it a complicated procedure (Jin et al., 2006). Silver staining is a method that offers great sensitivity and an easy to handle protocol, thus making it one of the most commonly used staining methods.

[1] Bio-rad.com, 'Coomassie Stains', 2015. [Online]. Available: http://www.bio-rad.com/webroot/web/images/lsr/products/electrophoresis/product_overlay_content/global/lsr_biosafe_coomasie_gel.jpg. [Accessed: 14- Jun- 2015].

However, there are limitations to using the silver staining technique. The primary issue with the technique is that the users lose the ability to use the color coded molecular weight marker as a reference post-staining. To combat this issue, researchers poke holes into the polyacrylamide gel so that they can retain their molecular weight ladder reference points. However, by doing this we ruin the integrity of the gel making the staining process much more likely to damage or destroy the fragile gel.

Source:http://labs.mmg.pitt.edu/gjoerup/silver_stain.jpg

Researchers who have used the technique in the past have also expressed concerns on the time it takes to develop the gel and stain; the possibility of “over-staining” the gel, and the generation of false positives due to the sensitivity of the technique. (An example would be the solutions reacting with particles in the air to provide an unneeded background on the gel)

Our goal is to develop a marker that, when interacting with the reagents used in the staining protocol, will stain in colour and in specific positions. This technology will aid in the identification of the protein(s) of interest post-staining. In order to do so, we will investigate how specific amino acids react with silver staining reagents. After determining specific motifs, we aim to create novel proteins that contain an excess of that motif, particular amino acid and/or chemical modifications that will generate a specific color after treating it with silver staining reagents. To obtain such proteins, we will introduce novel nucleotide sequences into a plasmid by in vitro transcription translation. E. coli cells with expression vectors will then be transformed with the new plasmids.

By testing different techniques to develop and stain the gels we hope to see if the expressed concerns are still an issue, namely time taken to develop the gels and stain them effectively. To investigate this we will use BioRad’s recommended protocol, our protocol developed in-house, and a technique documented in Electrophoresis.

With the current technique used today, researchers poke holes into the PA gel so that they can retain their molecular weight ladder reference points. Not only does this take time to do, but it also ruins the integrity of the gel making the staining process much more likely to damage the fragile gel.