Introduction
Antibodies are essential reagents in life science research—but using them incorrectly can lead to false results, wasted reagents, and frustration. Inconsistent data is often traced back to simple, avoidable mistakes like using the wrong antibody, skipping essential controls, or mishandling reagents.
At KinesisDx, we help scientists not only access high-quality antibodies but also use them effectively. This blog highlights the most common mistakes made when working with antibodies and how you can avoid them using scientifically proven best practices and technical guidance we follow at KinesisDx. searched questions, tips to improve performance, and reference-backed best practices.
1. Using an Antibody Not Validated for Your Application
The Mistake
Using an antibody in a workflow it hasn’t been validated for (e.g., using a WB-only antibody in IHC or ELISA).
The Risk
The antibody may not bind the target in its native form or under assay conditions, leading to no signal or incorrect results.
How to Avoid It
- Always check the validated applications listed on the datasheet.
- Look for image evidence in the relevant assay type.
- At KinesisDx, every antibody includes application-specific validation and data on supported protocols.
2. Ignoring Species Reactivity
The Mistake
Using an antibody against a protein in a species it wasn’t validated for.
The Risk
Even highly specific antibodies can fail or show cross-reactivity if used in an unvalidated species.
How to Avoid It
- Confirm species reactivity on the datasheet.
- Use sequence alignment (e.g., BLAST) to check for epitope homology.
- KinesisDx includes validated species and lists predicted cross-reactivity clearly.
3. Not Titrating Antibodies
The Mistake
Applying the manufacturer’s recommended dilution without optimization.
The Risk
Can result in weak signal or high background due to excess antibody.
How to Avoid It
- Perform titration experiments to find the optimal concentration.
- Use positive and negative controls to evaluate specificity and strength.
- KinesisDx protocols suggest titration ranges and provide example data curves.
4. Skipping Essential Controls
The Mistake
Not including controls like isotype, secondary-only, or KO samples.
The Risk
You won’t be able to determine whether observed signal is specific or artefactual.
Must-Have Controls
- No-primary control
- Isotype control (same species and isotype as your primary)
- Positive and negative controls (samples known to express or lack the target)
KinesisDx protocols always list the recommended control set by application.
5. Improper Blocking or Wash Buffers
The Mistake
Using inappropriate blocking agents or insufficient washing.
The Risk
Leads to high background, poor specificity, and inconsistent results.
Best Practices
- Use BSA, not milk, for phospho-antibodies.
- Wash 3–5 times with PBST (for ELISA/IHC) or TBST (for WB).
- KinesisDx datasheets suggest optimized buffers for each antibody.
6. Overexposing Signal
The Mistake
Over-developing chemiluminescent substrates or overexposing digital images.
The Risk
Creates oversaturation, “ghost bands,” or non-linear quantification.
How to Avoid It
- Perform serial exposures.
- Capture images within the linear detection range.
- Use a housekeeping control to confirm equal loading.
7. Improper Storage and Handling
The Mistake
Re-freezing multiple times, using non-sterile techniques, or storing at wrong temperatures.
The Risk
Loss of activity due to denaturation or microbial contamination.
KinesisDx Guidelines
- Aliquot antibodies upon first thaw.
- Store at –20°C or 4°C with glycerol (avoid frost-free freezers).
- Use only low-protein-binding tubes and sterile techniques.
8. Using the Wrong Secondary Antibody
The Mistake
Choosing a secondary antibody that doesn’t match the primary’s host or isotype.
The Risk
No signal or off-target background.
Best Practices
- Match host species and isotype exactly.
- Use cross-adsorbed secondaries to reduce species cross-reactivity.
- KinesisDx secondary antibodies are designed to pair with our primaries, with easy selection tools.
9. Misinterpreting Band Sizes
The Mistake
Assuming that any unexpected band is non-specific.
The Risk
Missing true isoforms, splice variants, or cleavage products.
What To Do
- Cross-check with UniProt for known isoforms.
- Use KO samples or peptide blocking for validation.
- Don’t dismiss an additional band until verified.
10. Applying One Antibody Across All Applications
The Mistake
Using the same antibody interchangeably across WB, IHC, ELISA, IP, and FC.
The Risk
Antibodies optimized for denatured proteins may not bind native epitopes, and vice versa.
KinesisDx Practice
We categorize antibodies by application and only promote them for validated use cases with data and protocol examples.
Additional Frequently Asked Questions (FAQ)
Why does my Western blot show multiple bands?
Possible reasons include:
- Non-specific binding
- Alternative splicing
- Post-translational modifications
Use knockout or peptide blocking to confirm specificity.
How many freeze–thaw cycles can my antibody handle?
Ideally none. Aliquot upon arrival to avoid degradation. Repeated freeze–thaw cycles degrade antibody structure and function.
Can I store diluted antibodies?
Not recommended unless using preservatives. Store diluted antibodies at 4°C and use within 1–2 weeks.
Why is my ELISA signal weak?
Could be due to:
- Incorrect antibody pairing
- Antigen degradation
- Poor coating conditions
Verify buffers, standard curve, and antibody concentrations.
What’s the best blocking agent?
- Milk for general use
- BSA or serum for phospho-antibodies
- For IHC/IF, use serum from the same species as the secondary host.
Why is IHC background high despite a specific antibody?
May result from:
- Endogenous Fc receptor binding
- Poor antigen retrieval
- Inadequate blocking
Use Fc-block, optimize retrieval, and titrate antibody concentrations.
How can I tell if my secondary antibody is causing background?
Use a secondary-only control. If signal remains, your secondary antibody is binding non-specifically.
Conclusion
Using antibodies effectively requires more than just picking the right catalog number—it requires understanding target biology, optimizing conditions, and validating results. Most experimental failures with antibodies are preventable.
At KinesisDx, we empower scientists with:
- Fully validated, application-specific antibodies
- Detailed datasheets with protocols and controls
- Supportive technical staff to troubleshoot with you
Whether you’re publishing or preparing a diagnostic assay, our antibodies are backed by rigorous testing to help you generate confident, reproducible results.
Visit www.kinesisdx.com to explore our antibody portfolio or access technical resources like our Troubleshooting Guide.
Works Cited
- Greenfield, Elizabeth A. Antibodies: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, 2014.
- Harlow, Ed, and David Lane. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1988.
Wikimedia Commons. “Immunodetection Troubleshooting.” https://commons.wikimedia.org/wiki/File:Immunodetection_troubleshooting.svg
