Antibodies in Practice: General Information

Selection and Use

Considerations when selecting an antibody for use in an experiment
Once you have identified your target antigen and have chosen your detection method, you must then choose one or more primary antibodies to detect your target. If more than one potential antibody is available for your target, it may be recommended to carry out key experiments using multiple antibodies (see Publishing with Antibodies). Choose your antibodies based on the following considerations:

Determine the best application for your research need:

  • Not all antibodies will work with every application.
  • Determine if you are performing a qualitative or quantitative assay.
  • Check vendor’s data sheet or website to see if the antibody is suitable for the specific application, such as immunoblotting, ELISA etc.

Type of sample being tested:

  • Does your tissue or cell express the particular protein?
  • Are you trying to detect a latent or activated protein? For example, phospho-specific antibodies may react only with activated phosphorylated proteins.
  • If your protein has an intracellular location it will be necessary to perform a cell lysis.
  • In flow cytometric analysis it may be necessary to use an antibody that recognizes cell surface molecules.
  • If your protein has a tertiary structure and the epitope is obscured then sample has to be denatured because antibody will not recognize the native state.
  • Some antibodies will work best only in frozen or on unfixed tissue and others will work in paraffin sections only after an antigen retrieval process.

Species from which the protein is to be detected:

  • Select an antibody that is raised against the immunogen sequence derived from species of your interest.
  • If the sequence is not derived from your species of interest, check to see if it will react with your sample. You may quickly check the sequence for specific proteins in the protein data bank: http://www.ncbi.nlm.nih.gov/protein.

Species in which the antibody is raised:

  • This information will be of great advantage when selecting a secondary antibody. The secondary antibody should be phylogenetically as far apart as possible from a species from which your sample is derived.

Check for validation data available on data sheet or vendor website:

  • Look at the validation data on data sheet or on vendor’s website and examine the quality of data.
  • Check to see if only a verification of the presence of antigen is provided (ELISA, Western blotting) or whether there are other in-depth data.
  • Check to see what type of sample was tested (cell lysate, tissue homogenate etc). Just using purified recombinant protein may not give best results with real cell or tissue samples.

Guarantee and support:

  • Is there an offer of guarantee from the vendor? It may be money-back or credit.
  • What type of technical support is available? It is best to have access to live technical support as opposed to frequently asked questions on the website.

Antibody Titer and Concentration

The binding of antibody and antigen is dependent on the affinity constant, which, in turn, can be affected by temperature, pH, solvent composition, etc. Varying the relative concentrations of antibody and antigen in solution can also control the extent of antibody-antigen complex formation.

Concentration and titer are not equivalent. Concentration is the total amount of antibody contained in the solution. Usually, only a percentage of it represents the intact, active, and functional antibody with regard to its ability to bind the antigen, and determines its effectiveness. The titer is the highest dilution of the antibody that yields a response in the immunoassay. It is the degree to which the antibody-serum solution can be diluted and still contain detectable amounts of antibody.

In most cases, the concentration of antigen in a sample cannot be adjusted. Hence, the optimal working concentration (dilution) of the antibody must be determined empirically for a given set of experimental conditions.

For any assay, the optimum titer is that concentration (dilution) which gives the strongest reaction for positive tests with minimum background reaction (e.g., for negative controls). The optimal antibody concentration must be determined experimentally for each assay, and is typically determined by using a dilution series.

The optimal antibody concentration is best determined by first selecting a fixed incubation time and preparing a series of dilutions to test. Dilutions are usually expressed as the ratio of the more concentrated stock solution to the total volume of the desired solution. For example, a 1:10 dilution of antibody is created by mixing one part of antibody stock solution with nine parts of diluent, giving a total of ten parts.

Datasheets and protocols may suggest approximate dilutions for antibody use. When using an antibody for the first time, or when working with a new batch of antibody, it is advisable to try a dilution series to determine the optimal antibody dilution to use. For example, if a product data sheet suggests using a 1:500 dilution, making dilutions of 1:50, 1:100, 1:500, 1:1,000 and 1:10,000 can help determine the optimal dilution for a set of unique assay conditions. Especially in the case of polyclonal antisera, antibody concentrations may be significantly different from animal to animal or from one serum bleed to the next, and this kind of initial titration is essential in reducing inter-assay variations.


Storage and Handling of Antibodies

The proper storage and handling of antibodies is critical to their function and longevity. Properly stored antibodies show little degradation over long periods of time, extending their usefulness to several months or even years. Improperly stored antibodies, on the other hand, can denature in a matter of hours. Consider the following points when storing and handling antibodies and other biological reagents:

  • In order to preserve maximum reactivity, reagents should be stored according to the manufacturer’s instructions (e.g., avoid holding antibodies at room temperature when storage at 2–8°C is indicated).
  • It is a good rule of thumb to store antibodies in tightly sealed containers in a non-frost-free refrigerator/freezer, away from tissue fixatives and crosslinking reagents.
  • Antibodies are relatively stable proteins and are resistant to a broad range of mild denaturing conditions. Most antibodies are stable for years when stored properly as per manufacturer’s recommendations.
  • In most cases antibodies can be stored at -20°C without any loss in their binding capacity.
  • It is best to avoid storing antibodies in a frost-free freezer. This is to avoid or minimize freeze-thaw cycles. Antibody solutions should not be frozen and thawed repeatedly, as this can lead to aggregation, causing a loss of activity. Hence, stock solutions should be aliquoted prior to storage.
  • Undiluted antibodies should always be aliquoted prior to storage at -20°C to minimize repeated freeze/thaw cycles that can denature antibody. Storing antibody in concentrated form will either prevent or minimize degradation. A cryoprotectant, such as glycerol, to a final concentration of 50%, can be added to the antibody solution to prevent freeze/thaw damage. Do not store glycerol-containing antibodies at -80°C. Unless a stabilizing protein, such as BSA (1% w/v), has been added, antibodies should not be stored for extended periods at their working dilutions. Avoid storing diluted antibodies for extended periods.
  • The major problem encountered during storage is contamination with bacteria or fungi. If antibodies are stored at 2-8°C for more than two to three days, it is advisable to filter-sterilization and/or add a bacteriostat/preservative, such as 0.05% sodium azide or 0.1% thimerosal.
  • Sodium azide can interfere with various biological assays and with some coupling methods. Hence, in these applications it is best to either remove sodium azide by centrifugal diafiltration, dialysis, or gel filtration, or use azide-free antibodies. Note : Sodium azide is toxic. As with all laboratory reagents, consult a Material Safety Data Sheet (MSDS) for handling precautions.
  • Generally, enzyme-conjugated antibodies are not frozen to prevent loss of enzyme activity and their binding capacity. It is best to store them at +4°C.
  • Fluorescent conjugates are susceptible to photobleaching. Hence, fluorochrome-conjugated antibodies should be stored protected from light in a darker colored vial.
  • When stored for a long period of time, some antibody solutions may produce an insoluble lipid component. The precipitate can be removed by a quick centrifugation at 10,000 g.