STAT3 Antibody

What are the major isoforms of STAT3 and why is this important for antibody selection?

STAT3 exists in multiple isoforms, with STAT3α (88 kDa) and STAT3β (79-83 kDa) being the most well-characterized. STAT3β is an alternatively spliced variant discovered 19 years ago in both mice and humans . When selecting antibodies, it's crucial to understand which isoform you need to detect:

• STAT3α is the full-length protein (770 amino acids in humans) and contains the canonical C-terminal transactivation domain

• STAT3β lacks 55 C-terminal amino acids of STAT3α but contains 7 unique C-terminal amino acids (CT7 epitope)

These isoforms exhibit distinct functional properties. Tyrosine-phosphorylated STAT3β homodimers are more stable, bind DNA more avidly, show prolonged nuclear retention, and are less susceptible to dephosphorylation compared to STAT3α homodimers . For accurate experimental interpretation, researchers must use antibodies that can either differentiate between these isoforms or detect both, depending on the research question.

What applications can STAT3 antibodies be used for?

STAT3 antibodies are versatile research tools applicable to numerous techniques:

Multiple search results demonstrate these applications. For example, anti-STAT3 antibody [EPR787Y] has been validated for Western blot, ICC/IF, flow cytometry, IHC-P, and ChIP/CUT&RUN applications . The Mouse Anti-Human/Mouse/Rat STAT3 Monoclonal Antibody (Clone 232209) has been validated for detecting STAT3 in immunocytochemistry and Western blot applications .

How can I validate the specificity of a STAT3 antibody?

Antibody validation is crucial for reliable results. Several approaches are recommended:

• Knockout cell lines: Use STAT3 knockout cell lines as negative controls. For example, the specificity of anti-STAT3 antibody [EPR787Y] was validated using STAT3 knockout HeLa cells, where the antibody detected STAT3 in wild-type cells but showed no signal in knockout cells .

• Cross-reactivity testing: Test antibodies against related STAT family members. The Mouse Anti-Human/Mouse/Rat STAT3 Monoclonal Antibody was validated by showing it detected STAT3 in parental HeLa cells but not in STAT3 knockout cells, while still detecting STAT3 in STAT1, STAT2, STAT5a, STAT5b, and STAT6 knockout HeLa lines .

• Overexpression systems: Use cells overexpressing tagged STAT3 proteins. In one study, GFP-tagged STAT3α and STAT3β were expressed in 293T cells to validate STAT3β-specific monoclonal antibodies .

• Multiple antibody comparison: Compare results from different antibodies targeting distinct epitopes of STAT3.

• Appropriate controls: Always include positive and negative controls in your experiments.

How can I specifically detect and differentiate STAT3β from STAT3α?

Distinguishing between STAT3 isoforms requires carefully selected antibodies targeting isoform-specific epitopes:

For STAT3β-specific detection, monoclonal antibodies recognizing the unique C-terminal 7 amino acids (CT7 epitope) have been developed. These antibodies do not cross-react with STAT3α or proteolytically cleaved STAT3 fragments . In validation studies, these antibodies could detect GFP-STAT3β at dilutions as low as 1:10,000 without any detection of GFP-STAT3α, even at high antibody concentrations (1:300) .

For STAT3α-specific detection, commercial antibodies like the STAT3α-specific monoclonal antibody (D1A5) specifically detect STAT3α but not STAT3β .

For total STAT3 detection (both isoforms), use antibodies targeting epitopes common to both isoforms, such as the N-terminal region or central domains.

When developing experimental approaches requiring isoform discrimination:

• Use Western blotting with both isoform-specific and total STAT3 antibodies

• Run appropriate positive controls (recombinant STAT3α and STAT3β proteins)

• Consider molecular weight differences (STAT3α at ~92 kDa, STAT3β at ~83 kDa)

What are the optimal methods for quantifying total and phosphorylated STAT3?

Accurate quantification of STAT3 requires calibrated immunoassays. A recommended approach involves:

• Immunoprecipitation of target proteins: Isolate total STAT3 or phosphorylated STAT3 from cell lysates using specific antibodies .

• Calibrator proteins: Use known amounts of recombinant unphosphorylated STAT3 calibrator proteins that share an epitope with your proteins of interest .

• SDS-PAGE and Western blot: Separate precipitated proteins alongside calibrator proteins, and detect using antibodies recognizing an epitope present in all protein species (STAT3, phospho-STAT3, and calibrator STAT3) .

• IP efficiency correction: Determine immunoprecipitation efficiency by comparing STAT3 levels in lysates before and after IP, then correct quantification accordingly .

This approach offers several advantages over standard Western blotting:

• Provides absolute quantities rather than relative values

• Accounts for immunoprecipitation efficiency

• Allows direct comparison between experiments

Remember that phospho-specific antibodies typically recognize specific phosphorylated residues (e.g., Tyr705 or Ser727), so be clear about which modification you're targeting.

How can I optimize STAT3 antibodies for immunoprecipitation studies?

For successful STAT3 immunoprecipitation:

• Antibody selection: Choose high-affinity antibodies validated for IP. Some antibodies work well for Western blot but poorly for IP due to epitope accessibility issues in native conditions.

• Lysis conditions: Use gentle lysis buffers that preserve protein-protein interactions. For phosphorylated STAT3, include phosphatase inhibitors to prevent dephosphorylation during lysis .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody amount optimization: Titrate antibody amounts - too little leads to incomplete precipitation, while too much can increase non-specific binding.

• Positive controls: Include positive controls to verify IP efficiency. The efficiency of IP can be monitored by comparing STAT3 levels in pre-IP and post-IP lysates .

• Negative controls: Include isotype-matched control antibodies (e.g., anti-STAT1 as a negative control for STAT3 IP) .

In a study developing nanobodies against STAT3, researchers successfully immunoprecipitated endogenous STAT3 from MDA-MB-231 cell lysates and demonstrated that their VHH-based antibodies showed higher efficiency than conventional antibodies .

What considerations should I make when studying phosphorylated STAT3?

Phosphorylated STAT3 detection requires special considerations:

• Rapid sample processing: Phosphorylation status can change quickly after cell lysis. Process samples rapidly and keep them cold.

• Phosphatase inhibitors: Always include phosphatase inhibitors in lysis buffers (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate).

• Phospho-specific antibodies: Use antibodies that specifically recognize phosphorylated residues. The most common for STAT3 are:

  • pTyr705 - Critical for dimerization, nuclear translocation, and DNA binding

  • pSer727 - Modulates transcriptional activity

• Positive controls: Include samples from cells treated with stimuli known to induce STAT3 phosphorylation (e.g., IL-6, IFNα, EGF) .

• Dephosphorylation controls: Consider including lambda phosphatase-treated samples as negative controls.

• Quantification methods: For accurate quantification, normalize phospho-STAT3 to total STAT3 rather than loading controls like GAPDH .

How can I troubleshoot non-specific binding or poor results with STAT3 antibodies?

When troubleshooting STAT3 antibody issues:

• Antibody validation: Confirm antibody specificity using knockout controls. Multiple sources show STAT3 antibodies that detect bands in wild-type cells but not in STAT3 knockout cells .

• Epitope accessibility: Consider different fixation/lysis methods if your epitope might be masked. For example, some epitopes are sensitive to certain fixatives.

• Blocking optimization: Test different blocking reagents (BSA, non-fat milk, serum) as some may contain phosphatases that affect phospho-STAT3 detection.

• Antibody dilution: Optimize antibody concentration. In one study, STAT3β-specific antibodies were effective at dilutions from 1:300 to 1:10,000, but optimization is experiment-specific .

• Signal enhancement: Consider using signal amplification methods for weak signals.

• Cross-reactivity: Test for cross-reactivity with other STAT family members. The Mouse Anti-Human/Mouse/Rat STAT3 Monoclonal Antibody was validated against multiple STAT knockout cell lines to confirm specificity .

• Secondary antibody optimization: Test different secondary antibodies; some secondary antibodies like Goat anti-Rabbit IgG H&L (IRDye® 800CW) and Goat anti-Mouse IgG H&L (IRDye® 680RD) have been successfully used for STAT3 detection .

What are emerging approaches for studying STAT3 in specific contexts?

Recent advances in STAT3 research include:

These approaches offer new insights into STAT3 biology beyond traditional Western blot and immunohistochemistry applications.