How to Choose the Right Antibody for Western Blot

How to Choose the Right Antibody for Western Blot

Introduction:
Western blotting remains one of the most widely used methods for protein detection and quantification in cell and molecular biology. However, it is also one of the most misinterpreted and variable techniques-largely due to inappropriate or poorly validated antibodies. A reliable Western blot begins with antibody selection based on precise parameters: validation method, target biology, clone type, application suitability, and detection sensitivity. In this guide, we’ll outline a research-grade approach to choosing the right antibody for Western blotting, drawing on examples from KO/KD-validated antibodies in the KinesisDx catalog.

Understanding the Requirements of Western Blot Antibodies

Western blotting involves protein separation under denaturing conditions via SDS-PAGE, transfer to a membrane, and immunodetection using a primary antibody. This means the antibody must:

Recognize a linear (denatured) epitope.

Bind with high affinity under reducing and non-reducing conditions.

Distinguish between isoforms or closely related proteins if necessary.

Perform in the context of total lysate, which includes many potential off-target proteins.

Therefore, specificity and validation against endogenous protein in denatured samples is essential—not just recombinant overexpression models.

Monoclonal vs Polyclonal in Western Blot Context

Monoclonal antibodies (mAbs) recognize a single epitope, reducing non-specific interactions. Ideal for detecting a single isoform or studying post-translational modifications.

Polyclonal antibodies (pAbs) recognize multiple epitopes on the same protein. Useful when the protein is low in abundance or subject to degradation but carry a higher risk of cross-reactivity.

Recombinant monoclonals combine consistency and specificity, minimizing batch-to-batch variability.

KinesisDx offers KO/KD-validated monoclonals and polyclonals optimized for WB, such as EGFR (KDXB200031) and CD147 (KDXB200225).

The Importance of KO/KD Validation in Western Blot

KO validation
uses CRISPR/Cas9-engineered cells lacking the gene of interest. If the antibody is specific, the band should be absent in KO lysates and present at the correct molecular weight in WT lysates.

KO validation
involves siRNA or shRNA-based knockdown, where a reduction (not complete loss) of band intensity confirms target specificity.

For example:
AKT1 (KDXB200156) shows complete signal absence in CRISPR-KO HeLa lysates at ~60 kDa.
STAT2 (KDXB200119) validated in siRNA-treated cells shows ~60–70% signal reduction at ~113 kDa.

This validation provides direct evidence that the antibody signal is not due to cross-reactivity with homologous proteins or nonspecific bands.

Considerations When Selecting a WB Antibody

1. Epitope Accessibility and Denaturation

The antibody must bind to a linear epitope exposed during SDS-PAGE. Some conformational antibodies (e.g., used in Flow or IF) may fail in WB.

2. Target Isoform and Post-Translational Modifications

Choose antibodies that distinguish between isoforms (e.g., EGFR vs EGFRvIII) or phosphorylation state (e.g., p-AKT1 vs total AKT1). Check the datasheet and KO blot to verify band specificity.

3. Host Species and Cross-reactivity

Ensure compatibility with your sample species. For human and mouse studies, cross-species validation (as available on KinesisDx datasheets) is ideal.

4. Molecular Weight Consistency

The observed band should match the predicted molecular weight ±5–10%. Consult UniProt and your datasheet.

5. Dilution and Blocking Optimization

Start at the recommended dilution (typically 1:500 to 1:2000) and optimize blocking buffer (5% BSA or non-fat dry milk) based on antibody formulation.

Examples of Validated Antibodies for WB from KinesisDx

Target	Catalog No.	Host	Type	MW	Validation
EGFR	KDXB200031	Rabbit	Monoclonal	~170 kDa	KO, WB, IHC
AKT1	KDXB200156	Rabbit	Monoclonal	~60 kDa	KO, WB, ELISA
STAT2	KDXB200119	Rabbit	Monoclonal	~113 kDa	KD, WB, IF
CD147 / BSG	KDXB200225	Rabbit	Polyclonal	~43–65 kDa	KO, WB, IHC
BetaIII-Tubulin	KDXB200134	Mouse	Monoclonal	~55 kDa	KD, WB, IF

Troubleshooting Antibody Performance in WB

If you observe unexpected bands or weak signals:

Check for isoforms or cleavage fragments.

Optimize lysis buffer and reduce degradation.

Confirm protein loading and transfer efficiency.

Compare your banding pattern to KO/KD validation images provided by the vendor.

Final Checklist for Selecting a Western Blot Antibody

The antibody is validated specifically for WB (not only IF or ELISA).

KO or KD validation data are available with clear comparison blots.

The predicted molecular weight matches your band.

The datasheet includes species reactivity and epitope type (linear vs conformational).

Clone type (monoclonal/polyclonal) suits your application need.

Signal strength and dilution recommendations are clear.

Representative blot images are shown in the datasheet.

Conclusion
Selecting the right antibody for Western blotting is not a matter of brand but of data. Use KO/KD validation, application-specific testing, and molecular weight alignment to choose reagents that deliver reproducible, biologically meaningful results. With its portfolio of KO/KD-validated antibodies, KinesisDx provides reliable reagents for WB detection of EGFR, AKT1, STAT2, and more.

VisitBrowse KO-Validated Western Blot AntibodiesWebsite

References
Uhlen M, Bandrowski A, Carr S, et al. A proposal for validation of antibodies. *Nat Methods*. 2016;13(10):823–827. doi:10.1038/nmeth.3995

Bordeaux J, Welsh AW, Agarwal S, et al. Antibody validation. Biotechniques*. 2010;48(3):197–209. doi:10.2144/000113382

Rimm DL. What brown cannot do for you. *Nat Biotechnol*. 2006;24(8):914–916. doi:10.1038/nbt0806-914