![]() Proteins must be transferred from the protein gel to an appropriate membrane (typically nitrocellulose or polyvinylidene difluoride (PVDF)) to facilitate antibody probing. However, this type of control can be problematic when comparing models in which “control” proteins are differently expressed, such as degeneration models. Detecting expression of a ubiquitous protein that should be even between all of your samples, such as actin in whole cell and cytoplasmic samples, can also be used as a loading control and helps to ensure consistent transfer of protein samples to the membrane. Running a duplicate protein gel and developing with Coomassie stain 5 can help to remove this uncertainty as it will show the amount of total protein 6 in each sample lane and can reveal any loading inconsistencies. This could mean that there is twice as much of the target protein in that sample, or it could mean that more sample or a more concentrated sample has been loaded in one lane than the other. For example, when assessing a blot, the band from one sample may appear twice as bright as another sample. It is essential, especially when trying to compare protein expression between different samples, to know how much sample has been loaded as this may not be apparent from the blot alone. It is also important to load appropriate control samples and size marker ladders to enable interpretation of the final blot. Solids will impair the running of the gel and it is likely your protein of interest will remain in the stacking gel. ![]() If your protein of interest is in the insoluble fraction (e.g., cell membrane-bound proteins) investigate pretreatment methods to liberate and solubilize it first. For a clean image, samples are centrifuged to remove any solids, in order to load only the soluble fraction. The specific separation method chosen will depend on the aim of the analysis. This is typically achieved by protein electrophoresis, such as sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or native PAGE, which separates proteins based on their molecular weight or charge. Therefore, consideration should be given to the host species in which the primary antibody was raised vs the sample species (e.g., if your sample is from a mouse, the primary antibody should not be from mouse since your secondary antibody would be directed against nonspecific mouse IgG).Figure 1: Overview of a western blot protocol.īefore a western blot can be performed, the proteins in the sample must be separated. They are usually directed against the IgG portion of the species in which the primary antibody was made. Secondary antibodies facilitate signal detection and amplification. Labeled antibodies against several important biological target proteins are commercially available. Since fluorophores with absorption and emission maxima spanning the entire light spectrum are available, combinations of antibodies conjugated to fluorophores of different wavelengths can be used to detect multiple proteins. By using different combinations of filters, specific wavelengths of light can be measured. These fluorophore-conjugated antibodies utilize the property of fluorophores to absorb light at a certain wavelength and emit it at a different wavelength.
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