purpose of secondary antibody in western blot

PURPOSE OF SECONDARY ANTIBODY IN WESTERN BLOT SERIES

Protein transfer to the membrane is essential because gels used for electrophoresis provide a poor surface for immunostaining, ie antibodies don’t stick to the proteins in the gel. The gel is then placed next to the membrane, and the use of an electrical current induces the proteins to migrate from the gel to the membrane. In the first step, the proteins are separated based on size by gel electrophoresis. To achieve this, western blot implements three steps: (1) separation by size, (2) transfer to a solid support, and (3) visualizing target protein using primary and secondary antibodies. Western blot allows us to determine the relative protein levels between samples and establish the molecular weight of the target, which can provide insight into its post-translational processing. Western blot, or western blotting, is a technique widely used in research to separate and identify proteins. You’ll get to grips with the essential information required before launching into the protocol itself.

PURPOSE OF SECONDARY ANTIBODY IN WESTERN BLOT SERIES

In Part 1 of our series on western blot, we’ll take you through the basics of this incredibly popular and useful technique. We’ll guide you through western blot basics and essential protocols before moving on to optimization, troubleshooting, and more advanced techniques. Traces of ß-mercaptoethanol will damage the antibodies.Welcome to our training series on western blot.Rinse the membrane under running water tap for 1–2 min.Dispose of the solution as required for ß-mercaptoethanol based buffers.Incubate at 50☌ for up to 45 min with some agitation Add the buffer to a small plastic box which has a tight lid use a volume that will cover the membrane.Prepare buffer and strip membranes under a fume hood.Īdd 0.8 mL ß-mercaptoethanol under the fume hood Repeat incubation for 5–10 min with fresh stripping buffer.Using a volume that will cover the membrane, incubate at room temperature for 5–10 min.If stripping is judged to be satisfactory, rinse the membrane several times with buffer, then block before proceeding to the antibody incubation.īring volume up to 1 L with distilled water Some researchers report successfully staining a membrane after stripping ten or more times.Įfficiency of stripping can be checked by incubating the membrane with chemiluminescent detection reagent. These steps can be repeated for probing with several antibodies, though the potential signal may be weaker and the background higher after each round of stripping. As a rule of thumb, try the gentler one first and then proceed to the harsher one if there is still a signal from the antibody that one is trying to strip. The following two protocols differ in harshness of treatment. Chemiluminescent reagents such as ECL are recommended as they will not leave a stain and are more sensitive than colorimetric reagents. Note also that colorimetric/chromogenic detection reagents will leave a permanent visible stain on the membrane that can interfere with subsequent detection of targets of similar molecular weights. For the same reason, a stripped membrane should not be probed to demonstrate the absence of a protein.Ī PVDF membrane is highly recommended to minimize loss of sample protein. It is not advisable to make quantitative comparisons of targets probed before and after stripping since the procedure removes some sample protein from the membrane. When probing for multiple targets, stripping and re-probing a single membrane instead of running and blotting multiple gels has the advantage of saving samples, materials, and time. Stripping is useful when one wants to investigate more than one protein on the same blot, for instance a protein of interest and a loading control. Stripping is the term used to describe the removal of primary and secondary antibodies from a western blot membrane. Our protocol for western blot membrane stripping and restaining includes step-by-step details on the removal of antibodies from western blot membranes.

Purpose of secondary antibody in western blot