# Calculating a Protein's Rf Value from a Western Blot

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:

 Rf = 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:

1. 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.
2. 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.
3. 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

St John’s Laboratory is your platform for purchasing commercially-validated and peer-reviewed primary and secondary antibodies.

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.

St John’s Laboratory believes the best way they can guarantee satisfaction in our products is through the provision of up to date testing data, as well as independent assessment - and thereby inviting all of their customers to take part in their Antibody Validation Project, where St John's Labs offer one of the greatest supplies of trial size antibodies available on the market.

St John’s Laboratory's vision is to give researcher partners as much control over their work as possible, by providing real up to date reviews and testing information. They encourage exploration of antibody specificity, species cross-reactivity and applications testing. They work alongside customers to carry out performance tests, so you can be assured that quality is the top of their agenda.

With St John's Laboratory platform, come and explore the performance of your required antibody before making a purchase and they’ll work to help you reduce the risk on your research, and save on essential research funds.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Nov 11, 2019 at 6:11 AM

## Citations

Please use one of the following formats to cite this article in your essay, paper or report:

• APA

St John’s Laboratory. (2019, November 11). Calculating a Protein's Rf Value from a Western Blot. News-Medical. Retrieved on January 26, 2020 from https://www.news-medical.net/whitepaper/20191111/Calculating-a-Proteins-Rf-Value-from-a-Western-Blot.aspx.

• MLA

St John’s Laboratory. "Calculating a Protein's Rf Value from a Western Blot". News-Medical. 26 January 2020. <https://www.news-medical.net/whitepaper/20191111/Calculating-a-Proteins-Rf-Value-from-a-Western-Blot.aspx>.

• Chicago

St John’s Laboratory. "Calculating a Protein's Rf Value from a Western Blot". News-Medical. https://www.news-medical.net/whitepaper/20191111/Calculating-a-Proteins-Rf-Value-from-a-Western-Blot.aspx. (accessed January 26, 2020).

• Harvard

St John’s Laboratory. 2019. Calculating a Protein's Rf Value from a Western Blot. News-Medical, viewed 26 January 2020, https://www.news-medical.net/whitepaper/20191111/Calculating-a-Proteins-Rf-Value-from-a-Western-Blot.aspx.