It should be noted that as the amount of acrylamide used increases the pore size decreases. Generally, for small proteins a high percentage gel should be used, for larger proteins a low percentage gel should be used. The percentage acrylamide used in the gel depends on the size of the protein of interest and size of pore needed in the gel. The basic chemistry of these two gels is identical they only differ in their acrylamide concentration and pH. The stacking gel is poured on top of the resolving gel – the combs are placed into this gel.
Once the resolving gel has set (allow up an hour) the stacking gel can be added. The bottom and larger portion of the gel is known as the resolving gel – this is poured first. There are 2 parts to an SDS-PAGE gel for a western blot. Once the sample has been lysed, the protein concentration determined and loading buffer added the sample can now be separated by gel electrophoresis. As the dye is anionic and small it will migrate the fastest of any component in the mixture to be separated and provide a migration front to monitor the separation progress. To enable visualization of the migration of proteins it is common to include small anionic dye molecule (e.g., bromophenol blue) in the loading buffer. Lameli buffer also has a SDS component which provides the negative charge necessary for gel electrophoresis, in addition to glycerol to make the sample denser. Lameli Buffer contains beta-2-mercaptoethanol or dithiothreitol (DTT) which act to reduce disulphide bonds before they adopt the random-coil configuration and in turn denatures the protein. The standard loading buffer for western blot samples is 2x Lameli Buffer. Protein denaturation can be achieved using a loading buffer containing a denaturing agent e.g SDS which is heated at 95-100☌ for 5 minutes Heat denaturation will unfold the protein and enable the antibody to bind its corresponding epitope located within the 3D conformation of the protein. Additionally, reducing agents such as ß-mercaptoethanol and DTT must be left out of the loading buffer and migration buffer.Ĭertain proteins will require denaturation for the antibody to work effectively. When this is the case it is not necessary to denature the sample and therefore SDS should be left out and the sample. In some cases, the antibody used will recognise the protein in native state, as the selected epitope may exist on the surface of the folded structure. This may be achieved by mechanical shearing. It is important to note that when preservation of protein-protein interactions is required a buffer without ionic and non-ionic detergents should be used. Information about the protein form your antibody recognises can be found on the data sheet supplied by the manufacturer. Lysis buffers containing SDS have a denaturing effect on protein, whilst buffers without detergent or mild non-ionic detergents such as NP-40 and Triton X-100 should be used when the antibody will only recognise protein in its native structure. It should also be noted that the lysis buffer used will affect antibody choice further on in the protocol with regards to the protein form it recognizes, either native or denatured. Lysis buffer containing sodium dodecyl sulfate (SDS) and other ionic detergents is considered to give the highest protein yield and is the most damaging to the sample. The choice of lysis buffer used will depend on the yield of protein required and the subcellular localisation of the protein.
The protein of interest must be solubilized in order to migrate through the separating gel.
The first step of a western blot protocol is protein extraction from cells or tissue.
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