![]() Protein A-HRP can be used for normal western blots as well, and would replace secondary antibodies against Mouse, Rabbit, and Guinea Pig, which might be an attractive option for labs on a budget or with a fair number of undergraduates, who seem inexplicably drawn to the incorrect secondary antibody. This system has (seemingly) been incorporated into several kits, but Protein A-HRP alone is also available from a number of companies. This means that it binds tightly to the primary antibody used in the western, but largely ignores the denatured antibody stuck to the membrane. Unlike a secondary antibody, Protein A only recognizes correctly folded antibodies. Protein G worked well, also, but did show higher affinity to the denatured antibody. However, in 2005 Ashish Lal published a method to circumvent this problem: instead of using a secondary antibody to detect the location of the primary, use HRP conjugated Protein A instead. If your immunoprecipitation (IP) used an antibody from the same organism as the primary antibody for your western blot, then your secondary antibody will not only recognize the primary bound to the epitope, but also the antibody from the IP that was denatured and separated out on the gel, creating giant background bands around 55 and 25 kD on the blot. Use Protein A-HRP to Get Clean Western Blots of Immunoprecipitated Samples These parameters are going to vary for each set of antibodies and sample types that one uses, so you would need to decide if it is worth you time to systematically optimize these incubation times. She could develop a blot and be ready for exposure within 45 minutes of removing the membrane from transfer. She found that the blot was blocked after a 10 minute incubation with fresh, room temperature 5% milk, and that the western signal reached maximum after a 15 minute incubation with her antibodies (without increasing the concentration of the antibodies). A friend of mine experimented with the blocking time and incubation time with the primary/secondary antibody solution. If you find yourself doing the same western blot repeatedly (i.e.same primary and secondary antibodies with similar samples), then you might be able to shave even more time off the standard protocol. I have used this method for around five years, and have yet to have any problems with it. For the average user, this will shave over an hour off a western blot by eliminating the second antibody incubation along with the washes between the two antibody incubations. Both the primary and secondary antibodies are diluted into your favorite solution (TBST + 5% milk, for example) and incubated with the blocked membrane at the same time. However, since the primary antibody binds its epitope with its variable domain and the secondary antibody with its constant domain, there is no reason that mixing the two antibodies together will interfere with the binding of the primary antibody to its epitope. Normally, we incubate the blocked membrane with the primary antibody, wash away the unbound antibody, then incubate the membrane with the secondary antibody which binds to the primary antibody, co-localizing the detection method (fluorophores, horseradish peroxidase (HRP), or alkaline phosphatase (AP)) with the protein of interest on the membrane. There are a number of kits coming out which, I believe, are based on this simple concept. Save Time by Co-Incubating the Primary and Secondary Antibodies However, there are some new products that are available and some tweaks that can be made to the protocols that may improve your results and reduce the time it takes you to execute this popular technique. Western Blotting is a long established method for which the protocol varies little from lab to lab. ![]()
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