Antibody valitation: Practical guide to finding and validating suitable antibodies for research
Step 1. Define your initial requirements
The starting point when sourcing antibodies is to correctly identify your target protein. Having the complete information on the target antigen (approved nomenclature, protein sequence, splicing variants, post-translational modifications, proteolytic cleavage and sequence homology of the target protein) is key to determine how specific of your target protein can be your antibody. Unfortunately, and for different reason, this information is not always available. But if available, it is well worth performing some basic bioinformatics analysis of the target antigen to address some important questions:
- Is the immnunogen used unique for the target protein (sequence homology)?
- Can my antibody recognize different isoforms of the same protein?
- Can my antibody recognize other family members?
You should also evaluate which type of antibody may be the best reagent for your research, and for which application you are planning to use it.
To define your initial requirement you should:
- Step 1: Define your initial requirements
- 1.A Identify the target antigen
- 1.B Define antibody requirements
- 1.C Decide on the experimental techniques you wish to use
- Example step 1
- Step2: Collect information and find existing antibodies
- 2.A Review the published peer-reviewed literature
- 2.B Find existing antibodies
- 2.C Review product information
- 2.D Match antibody data to existing information
- 2.E Study the individual company catalogues
- 2.F Accurately identify individual antibodies
- Example step 2
- Step 3: Assess existing validation data and decide whether further validation is necessary
- 3.A Reactivity with the target antigen
- 3.B Its suitability for use in your intended applications
- 3.C Decide whether further validation is necessary
- Example step 3
- Step 4: Reproducibility and dissemination of data
- Positive and negative controls for antibody validation
- Articles about antibody validation