A western blot is a popular tool for quantifying, determining, and identifying the size of particular proteins. Western blotting enables the separation of a mixture of proteins utilizing gel electrophoresis.
The proteins are then transported to a membrane from the gel (the ‘blotting’ step). The protein of interest is established by washing the membrane with a solution comprising of a primary antibody.
A secondary antibody that can be visualized (through immunofluorescence or staining) is introduced. If successful, the result provides a distinct image with defined bands displaying the target protein.
Calculating the Rf Value
The ratio of the distance moved by the solute to the distance moved by the solvent is the value of the Rf (retardation factor). The term was founded in chromatography, where it was noted that a specific compound will always travel a consistent distance in a particular solvent, so long as the conditions are stable.
Calculating the Rf value in western blot is beneficial as it enables the molecular weight of a protein to be established. The formula is:
||Migration distance of the protein
||Migration distance of the dye front
To calculate the Rf value by hand (instead of using software), these simple steps should be followed:
- In millimeters, measure the distance that the protein of interest has traveled. Ensure that the image position is known so that the correct distance is measured. As an example, if the anode (+) was at the bottom of the gel, the negatively-charged proteins will have traveled from the top to the bottom of the gel.
- Calculate the migration distance of the dye front, for example, how far the dye has traveled from the top of the gel. When this has been calculated once, it can be used to determine Rf values of various proteins in the sample.
- The formula above can then be utilized to determine the Rf value. The top value will always be bigger than the value at the bottom value so that the result will be less than 1.
Calculating Rf is relatively easy as seen in the above steps. The Rf values from the western blot may then be used to establish the molecular weight of the protein of interest. In the next article, this process will be outlined.
About St John's Laboratory
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Once established in 2013, St John's Labs knew that antibody validation was rapidly becoming a widespread concern for bioscience. In acknowledgement of these concerns, they invited a collective of industry experts, and business leaders to join the world’s First Antibody Validation Forum in 2014. See clips of the discussions which took place on their blog pages, or YouTube channel.
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