Translatome: The proteins expressed under certain conditions.
Polyacrylamide gel electrophoresis (PAGE) is the technique used to separate proteins. They are boiled with SDS page to denature them and coat them with negatively charged SDS which allows them to all move through the electric field uniformly despite having different amino groups on them. The only factor affecting their movement through the gel will now be size. After PAGE the proteins are dyed to be visualised.
2D PAGE can be used to further enhance the resolution of protein separation. Firstly proteins can be separated by isoelectric focussing, which separates proteins based on their inherent charge which comes from the amino acid residues in their structure by running them against a pH gradient. After this is done, the proteins are coated in SDS and an SDS-PAGE is run orthogonally to the isoelectric focussing. 2D-PAGE can resolve 1000 different proteins from E. coli. The protein of interest on the gel can then be cut out, digested and analysed in a mass spectrometer.
Three samples can be run using differential gel electrophoresis (DIGE) by staining each sample with a different fluorescent dye. 2D page has trouble identifying giant proteins which do not enter the gel or tiny fragments which run off the end of the gel. Rare proteins within the cell will hardly be visible and overrepresented proteins can interfere with other proteins in the gel because they saturate their local environment, and finally one has to remove the acrylamide after 2D-PAGE to analyse their sample in a mass spectrometer.
Before one can perform a western blot they need to have run a 2D-PAGE or a normal SDS-PAGE, and they need antibodies to the protein of interest. In the western blot, proteins are transferred to nitrocellulose membranse from the gel under an electric field. Nitrocellulose and other membranes are positively charged and areas of the membrane with no protein bound to them (because there was none in that location of the gel) will remain positively charged. These are blocked using milk proteins so that the antibody doesn’t non-specifically bind to these blank areas. The antibody is then added in solution over the nitrocellulose and given time to bind to the protein of interest. The target protein has an epitope specific to the antibody chosen. Next a secondary antibody which binds non-specifically to the primary antibody is added. The secondary antibody will have some sort of tag which will allow its location to be ascertained. Horseradish peroxidase is a common tag which oxidises luminol when in the presence of H2O2, emitting a pulse of light. A CCD is placed above the membrane to visualise the location of the protein. Photographic film can also be used. Western blots are the “bread and butter” of protein analysis.
High performance liquid chromatography (HPLC) is able to separate mixtures of proteins. Proteins are dissolved into the mobile phase and forced under high pressure through a narrow column. As the mixture elutes, the proteins separate as they will have different binding affinities to the column. Different detectors can be used in HPLC to detect proteins a they elute off the column, such as fluorescence detectors, refractive index detectors, UV detectors, radiochemical detectors or electrochemical detectors. Typically the proteins are treated with phosphatase so that separation efficiency is increased.
Size exclusion chromatography: the principle behind this is the use of porous beads that separate molecules based on size. Large molecules do not diffuse well into the pores and therefore have a lower residence time within each bead, whereas small molecules will diffuse into beads and spend some time there meaning they elute slower.
Reverse phase HPLC: This relies on the hydrophobicity of the stationary phase. Hydrophobic molecules will bind with more affinity to the stationary phase, thus eluting slower.
Ion exchange HPLC: The stationary phase is charged with functional groups which attract molecules with opposite charges. Any molecules which stick to the stationary phase will remain there. The buffer can be used to change the pH to change the next charge on proteins to get them to attach/detach from the stationary phase.
Affinity HPLC: uses a molecule on the stationary phase which binds a specific target. Most commonly this is an antibody. These are commonly used in the pharma industry for purifcation of monoclonal antibodies. Protein A columns are very common (and expensive).
Good separation means achieving a high resolution. Peaks should be narrow and symmetrical so that it’s easy to integrate the peak to determine the relative amount of protein.
MALDI (matrix-assisted laser desorption-ionisation) is a mass spec technology that allows for protein analysis. The peptides from the protein are first embedded into a solid matrix before they are ionised. Then the matrix/peptide mixture is ionised with a laser and the resulting ion fragments travel through a time-of-flight (TOF) tube. At the end of the TOF tube is a detector which reads the intensity of the ion hitting it and calculates the mass of the ion. The time spent in the tube, or the time of slight is proportional to the mass over charge ratio.
References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.