In the day-to-day pressures of a research environment, it is natural to want to generate important data as quickly as possible. However, there are real benefits in taking the time to carefully select, aliquot, and titrate your antibodies. Bio-Rad recently launched a free Antibody Advice Guide to equip scientists with the knowledge to get the best out of their experiments with antibodies. This article highlights five examples of valuable information that you will learn from the guide, helping you generate publication-ready data.
Consider clonality when choosing an antibody.
The product information for most antibodies will mention whether they are monoclonal or polyclonal. It’s easy to overlook this detail, assuming that any antibody that recognizes your target protein will be a good choice. However, monoclonal and polyclonal antibodies have different strengths and weaknesses, so it’s worth investing time in deciding which one to use.
First, it’s important to understand the difference between monoclonal and polyclonal antibodies. The specific part of an antigen recognized by an antibody is called the epitope. A monoclonal antibody recognizes only a single epitope on the target protein, whereas a polyclonal antibody recognizes multiple epitopes. This difference originates from the antibody production method. Each monoclonal antibody is produced from expansion of a clonal population of B cells, whereas a polyclonal antibody represents a mixture of antibodies from a nonclonal B-cell population. For this reason, monoclonal antibodies have clone names, whereas polyclonal antibodies do not.
If you need to detect a specific epitope on your protein, choose a monoclonal antibody which states that it detects that epitope. This can usually be found under “immunogen” in the product information. If you just want to detect the whole protein, regardless of the binding site, a polyclonal antibody may be appropriate.
The Antibody Advice Guide covers other things to consider when choosing an antibody, such as format and formulation, and whether an antibody has been validated using relevant methods. To learn about the importance of validated antibodies for trustworthy data, watch our short video.
Think about your epitope location.
Your target protein, and therefore your target antigen and its epitope, may be located on the cell surface, in the cytoplasm, nucleus, or elsewhere, depending on the protein and biological status. For specific detection, remember that antibodies are large proteins which may not easily pass through intact membranes.
Therefore, the location of your target epitope should influence your experimental design. If you are using whole cells rather than a lysate, detection of an intracellular epitope will require fixation and permeabilization to give the antibody access. It may be important to avoid damaging the cell membrane while doing this.
To complicate things further, membrane-bound proteins may have intracellular and extracellular epitopes. Whether or not permeabilization is required will depend on the exact location targeted by your antibody.
Download the guide to learn more about how your experiments should be guided by epitope location. You can also learn about antibodies that require specific buffers, such as antibodies used for cytokine staining or functional assays.
Aliquot your antibodies.
Antibodies are proteins that can degrade if they experience too many freeze-thaw cycles, so correct storage and handling are essential for experimental success. While it can be tempting to throw your new antibodies in a freezer box and thaw them every time you need to use them, some simple best practices can maintain the quality of the antibody to avoid wasting time and money.
When you receive an antibody, it’s a good idea to aliquot it into smaller quantities, with each vial containing the amount that you’ll use for an experiment. Aliquoting also makes it easy to see how much of an antibody you have left in your supply, avoiding moments of frustration when finding out last minute that you don’t have enough for an experiment.
To aliquot an antibody, briefly spin down the contents and then distribute into low protein‒binding tubes. Make sure to have at least 10 µl in each tube to avoid evaporation and concentration changes. Keep antibodies at their original concentration rather than diluting them; antibodies at lower concentrations have a greater chance of degrading or sticking to tube walls.
The Antibody Advice Guide will teach you valuable best practices for handling and storing antibodies, such as the best places in the freezer to keep them, and why frost-free freezers should be avoided, ensuring that you don’t waste time using damaged or degraded antibodies.
Titrate for optimal staining.
Do you just use antibodies at the manufacturer’s recommended concentration, or do you work out what’s best for your own experiments? A little optimization can set you up for long-term success.
Using too little antibody will result in low detection, but too much can lead to background staining from nonspecific binding and will also waste your antibody. Titrating an antibody involves testing it at a range of concentrations or dilutions, finding the concentration that generates the best signal-to-noise ratio in your individual application.
Specific titration guidance is available for western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The guide will teach you how to titrate antibodies for these applications, preparing you for your experiments.
Choose the right controls.
Appropriate controls are essential to validate your data and ensure that you can interpret your results correctly, proving that your experiment is able to detect true positive and negative results. It can be tempting to bypass controls, considering them to be a waste of resources, but they are crucial for trustworthy data.
All experiments using antibodies should include some essential controls:
- Positive and negative controls: showing that your experiment is able to produce and detect positive and negative results
- Secondary-only controls: to confirm that a secondary antibody, if used, is not producing false-positive results
- Unstained controls: for flow cytometry, fluorescent immunohistochemistry, and immunocytochemistry, an unstained control will ensure you are not seeing a false-positive result and helps to determine background fluorescence
Specific controls are needed for different applications. Download the Antibody Advice Guide to learn which controls are needed for western blotting, flow cytometry, ELISA, and immunohistochemistry.
For example, are you aware of Fc block controls for flow cytometry? The addition of reagents that block the Fc receptors on immune cells can help prevent false-positive results.
Antibodies are sometimes overlooked but investing some time in understanding their biology will help you generate cleaner data more efficiently, reducing time wasted on failed experiments. Download the Antibody Advice Guide and keep it accessible in your lab so that it is available for consultation.