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Tips for Choosing and Using a New Primary Antibody

primary antibody

Finding a good primary antibody can often feel like playing Russian roulette.  Nothing is more disappointing than buying a $300 antibody that doesn’t work for your use.

There are some steps you can take, however, to increase your likelihood of success.

Scout out Other Labs

Before you buy, ask if anyone around you or in your department has an antibody that you could test out in your application.   Some departments have a department-wide email list that is great for this purpose.  Ask for an antibody to the protein you are interested and tell them what you are going to use it for.  Make sure you are specific about desired species specificity if it is important for your experiment.

If someone does give you an antibody, make sure they give you all the information you need to get more if it does work for you.  An antibody of unknown origin is no good for future experiments.  You don’t want to have to do the antibody search over again.  I learned this lesson the hard way!

Down the road, don’t forget to acknowledge their help in any talks or papers.  A little lab PR goes a long way.

Take the Time to Do Research and Compare

If you do strike out on your own to find a primary antibody, use the Internet to generate a list of companies that market antibodies that recognize your protein of interest.  Carefully note the name of the antibody, particularly if it is from monoclonal antibody production.  I have seen the same monoclonal antibody, using in vitro monoclonal antibody production, sold by different companies for different prices.

Also, make sure you compare aliquot sizes, prices and recommended dilutions for use.  Some companies sell larger-sized, less concentrated aliquots.  In this case, larger doesn’t mean more.

Find the Right Antibody for the Job

When scouting the companies, make sure you find an antibody that fits your use.

Are you doing immunohistochemistry or fluorescent microscopy? Use the Internet to find images showing use of potential antibodies.  You can find images in publications but sometimes companies post images on their site.

Read the details carefully to make sure the antibody fits your application.  For example, for Western blots, many companies show images generated from transfected cells that overexpress the protein of interest.  You might get in the lab and discover the antibody is not sensitive enough to detect protein expressed at a physiological level.

If you are doing ELISA, look for ELISA-specific paired antibodies matched by species and antibody structure to give the best results.

Or maybe you are interested in monoclonal antibody therapy?  Then you better make sure the antibody is safe for in vivo use!

Don’t forget to determine if you need your antibody conjugated directly to a label or tag.  Primary antibodies can be conjugated to fluorescent dyes, affinity tags and reporter enzymes.  Direct conjugation means you won’t also be searching for the perfect secondary antibody.

Talk to the Company

Once you have decided on a potential antibody or 2 , call the company that sells the antibody and talk to technical services. Ask about their production of monoclonal antibodies (or polyclonal ones).  Find out if they can send you a protocol they have used successfully with the antibody.  Ask if they can send a sample size or free aliquot to try out the antibody (this works particularly well if you cannot find documented evidence of your intended use).  Ask if they have a return policy if the antibody doesn’t work for you.

Optimize the Primary Antibody for Your Use

When I get a new primary antibody I am always anxious to jump right in and try it out in a “real” experiment.  Invariably this is a bad idea!  Take the time to do a preliminary experiment.  Use positive and negative controls if you have them.  Make sure you try out the antibody at several different concentrations.

What are your tips for finding and using new primary antibodies?

Image Credit: woodleywonderworks

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