Antibody valitation: Practical guide to finding and validating suitable antibodies for research
2.A Review the published peer-reviewed literature
1. Look for existing antibodies and protein expression data: For many targets there will already be widely cited, well-characterised reagents. The main websites for this are listed below:
PubMed: This is the best route to identify antibodies that are well characterised and widely used.
CiteAb: An antibody search engine which ranks antibodies by citations.
BenchSci: The goal of Benchsci is to help scientists find antibodies that have been proven to work by the scientific community, and therefore achieve scientific discoveries faster.
Search Engines: e.g. Google This website analyses a wider range of documents and searches their entire content. Try using ‘antibody’ and the name of the target antigen.
2. No protein expression data is found in the literature: If no mAb against the antigen of interest has been previously described in the literature, you will need to seek as much information as possible related to its expression at the transcript level. In most cases this information in both normal human and murine tissues and in human tumours can be obtained from the databases listed below. Although transcript data can be useful to assess the likely distribution of a protein, it should be remembered that post-transcriptional regulation does not guarantee that there will be target protein actually expressed in cells and tissues. There is also emerging data from proteomic studies that can, however, be used to identify tissues expressing your antigen. Some useful links for transcript and proteomic data are provided below.
BioGPS: Transcript expression from microarray data.
GeoProfiles: Transcript data.
Oncomine: Transcript data.
Cbioportal: RNA sequence data from cancer tissues and cell lines.
- 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