“Is it the antibody?” Tips for caring for your antibody for consistent results

Since antibodies are so critical to your research, why not provide them with the best care possible so they can continue to work hard for you. 

Probably the worst thing to happen to a researcher is to have an experiment work once and then never work again. There are lots of possible reasons why such a scenario could occur, however one question that comes up time and time again is: “is it the antibody?”  Antibodies are generally robust, but they still need proper care in order to perform consistently. We have over 35 years of experience with antibodies, and here we provide our expert advice on what you can do in your lab, before starting your experiment, to facilitate the success of your antibodies.

What to do right away

Caring for your antibody starts the minute you receive it. 

• The first thing we recommend is that you check the accompanying technical data sheet (TDS). Think of this as the manual that comes with your new gadget. Certainly you would read this first before using the device right? Well so is the case with the TDS, consider it the manual for your antibody. It  contains useful information on how the antibody should be stored, the applications for which it is recommended, appropriate dilutions/concentrations for various applications, as well as references highlighting where the antibody has been used. Reviewing this information upfront will let you know what the best suggested practices are for the antibody and could save you lots of troubleshooting time. 

• You will likely next be ready to store your antibody based on the recommendation on the TDS (more information on antibody storage below). However, before storing, we suggest inverting the antibody and centrifuging briefly at low speed to collect any liquid that might be on the sides of the vial or in the cap.

• For antibodies intended for long term use, it is important to aliquot the antibody at a range between 10 to 50 µl in low protein binding microcentrifuge tubes. Be sure to store antibody aliquots in the concentrated form, as storing diluted antibodies decreases their effectiveness. 

Ensure proper storage conditions

Antibody shelf life depends on the nature of the antibody and the storage conditions. Long term storage at room temperature can lead to degradation or inactivity, usually as a result of bacterial growth. 

• Choose the right storage temperature for the antibody. Most antibodies can be stored long term at temperatures below zero (-20°C or -80°C). However storage may depend on frequency of use. For instance, if you plan on using your antibody 1-2 times per week, then it is safe to store it at 4°C throughout the course of your experiment. Enzyme-conjugated, RPE-conjugated, IgG3 isotype and IgM antibodies should be stored at 4°C, and should not be frozen. 

• Avoid freeze/thaw damage. As you may already know, repeated freeze/thaw can denature the antibody. However you may not know that the freezer the antibody is stored in can also affect its stability. The freezer used should NOT be the frost-free kind, as these freezers cycle between freezing and thawing, which can damage your antibody. 

Test under your unique experimental conditions before the real experiment

It is good laboratory practice to optimize your antibody for your needs prior to starting your experiments. This may not be the fastest way to results but it is certainly the best way to consistent results. 

• Determine the optimal antibody concentration for your samples. The TDS will provide a recommended concentration at which the antibody should work in the majority of cases. However, if you have unique experimental conditions, then it is best to confirm this by titrating the antibody, perhaps using the recommended concentration as a starting point. This should ideally be performed on your planned experimental samples.  Each new batch of antibody should be retested prior to use. 

• Confirm suitability for your intended application. Antibodies are tested for specific applications before being sold but the same antibody may also work for other applications. If you are using an antibody for an application for which it has not been recommended, then extensive testing will be required. Be sure to include appropriate controls, specifically negative controls such as a sample that does not express your target protein.