Introduction
An antibody datasheet is not just a label—it’s a roadmap to successful experiments. Yet many researchers overlook important details when selecting or troubleshooting antibodies. Knowing how to interpret antibody datasheets can prevent costly errors, reduce experimental variability, and ensure you’re using the right reagent for your assay.
This blog explains each section of a typical antibody datasheet and what to look for when selecting reagents for Western blot, ELISA, IHC, or flow cytometry.
1. Antibody Name and Catalog Number
Every datasheet starts with a catalog number, product name, and clone ID (for monoclonal antibodies). These identifiers are essential for:
- Tracking batch usage
- Reordering the exact same product
- Referencing in publications
If you’re using a monoclonal antibody, ensure you cite the clone ID in your methods section.
2. Host Species
The host species refers to the animal in which the antibody was raised (e.g., rabbit, mouse, goat).
Why it matters:
- It determines which secondary antibody you’ll need.
- If working with tissues from the same species (e.g., using a mouse antibody on mouse tissue), you risk high background due to cross-reactivity. In that case, consider species-specific blocking or using antibodies from a different host.
3. Reactivity / Target Species
This section tells you which species the antibody is known to recognize (e.g., human, mouse, rat). It’s determined through:
- Sequence homology
- Empirical testing (e.g., WB with species-specific lysates)
Tip: If the datasheet lists “predicted to react with,” validation may be lacking—test it cautiously or contact the supplier for evidence.
4. Clonality: Monoclonal vs Polyclonal
- Monoclonal antibodies bind to a single epitope; high specificity and reproducibility.
- Polyclonal antibodies recognize multiple epitopes; often more sensitive but may vary between batches.
Check if your application requires high specificity (monoclonal) or higher signal amplification (polyclonal).
5. Isotype
The isotype refers to the class of the antibody (e.g., IgG1, IgG2a, IgM). It affects:
- Fc-mediated functions (important in therapeutic and immune assays)
- Selection of appropriate secondary antibodies
- Background staining in assays like IHC and flow cytometry
Understanding isotypes is essential in multiplex assays where different isotype-specific secondaries are needed.
6. Applications
A key part of any datasheet is the list of validated applications:
- WB = Western blot
- IHC = Immunohistochemistry
- ICC = Immunocytochemistry
- ELISA = Enzyme-linked immunosorbent assay
- FC/Flow = Flow cytometry
- IP = Immunoprecipitation
Each application has unique requirements for antibody affinity, specificity, and epitope accessibility. Use only antibodies validated for your intended application.
Note: “Suggested applications” may not be validated—refer to publications or technical support for evidence.
7. Dilution Recommendations
Datasheets typically suggest a range for dilution (e.g., 1:500 to 1:2000). These are:
- Starting points—optimize in your system
- Affected by antigen abundance, detection method, and antibody affinity
Pro Tip: Perform a checkerboard titration when using an antibody for the first time.
8. Immunogen / Epitope Information
This tells you what the antibody was raised against:
- A full-length protein
- A recombinant fragment
- A synthetic peptide (with exact amino acid sequence)
Why it matters:
- Helps you predict cross-reactivity
- Tells you whether the antibody may bind denatured vs native forms
- Useful for epitope mapping and selecting controls
9. Validation and Specificity Data
Reputable suppliers provide:
- Western blots showing band size
- IHC images with positive and negative tissues
- Knock-out or knock-down validation
- Peptide blocking experiments
If a datasheet lacks validation images, request them. A robust datasheet often includes links to peer-reviewed publications using that antibody.
10. Purification and Buffer
Check how the antibody was purified:
- Protein A/G affinity
- Antigen-affinity
- Crude serum or ascites (less desirable)
Also, review the buffer formulation:
- Sodium azide: antimicrobial, but toxic to cells
- BSA, gelatin: used as stabilizers, but may interfere with conjugation or downstream applications
- Glycerol: allows freezing at –20°C
11. Storage Conditions
Always read the storage instructions:
- Short term: 2–8°C
- Long term: aliquot and store at –20°C or –80°C
- Avoid repeated freeze–thaw cycles
Proper storage ensures antibody stability, especially for long-term experiments or diagnostics.
12. Troubleshooting Notes
Some datasheets include application-specific troubleshooting tips, such as:
- Use of BSA over milk for phospho-antibodies
- Preferred antigen retrieval method in IHC
- Optimal incubation times and temperatures
Frequently Asked Questions
What is an antibody isotype and why does it matter?
Isotype refers to the class (IgG, IgA, etc.) and subclass (IgG1, IgG2a, etc.) of the antibody. It affects interactions with secondary antibodies and effector functions.
How do I know if an antibody is validated for Western blot or IHC?
Look for specific validation data in the datasheet—image evidence, band size, and referenced publications.
What should I do if my antibody is giving non-specific results?
Check if the application is validated, verify dilution, block nonspecific binding, and confirm species reactivity.
Conclusion
A well-structured antibody datasheet contains all the essential information to design, execute, and troubleshoot experiments successfully. By understanding each section—host species, applications, clonality, isotype, immunogen, and validation—you can select the best antibody for your needs and avoid unnecessary delays.
At KinesisDx, we aim to provide transparent, scientifically detailed datasheets alongside high-quality antibodies to support your research with precision and confidence.
Works Cited (MLA Format)
Thermo Fisher Scientific. “How to Read an Antibody Datasheet.” Technical Resources, 2024, https://www.thermofisher.com.er.com
Harlow, Ed, and David Lane. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1988.
Greenfield, Elizabeth A. Antibodies: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, 2014.
Bordeaux, John, et al. “Antibody Validation.” BioTechniques, vol. 48, no. 3, 2010, pp. 197–209.
Uhlén, Mathias, et al. “A Proposal for Validation of Antibodies.” Nature Methods, vol. 13, 2016, pp. 823–827.