Exceptionally Clean LEF1 Antibody

September 29, 2026

BlueskyReddit
Biochemistry
Stanford University
Senior Research Scientist

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Company:

Cell Signaling

Product Name:

LEF1 (C12A5) Rabbit Monoclonal Antibody

Catalog Number:

2230

Clone Number:

C12A5

Info:View Product Specs

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WNT signaling is essential for embryonic development and adult tissue homeostasis, and its aberrant activation drives several cancers, most notably colorectal cancer. My lab studies the molecular mechanisms of oncogenic WNT signal transduction, with the goal of identifying therapeutic vulnerabilities in colorectal cancer. Stabilized β-catenin acts in the nucleus with TCF/LEF transcription factors, so we use subcellular fractionation to follow pathway components across compartments during WNT stimulation.

Experimental Design and Results Summary

Applications

Western Blot

Sample

HEK293T cytoplasmic and nuclear fractions

Primary Incubation

1:1000

Blocking Agent

5% milk in TBS-T (0.1% Tween-20)

Secondary Incubation

HRP-conjugated Donkey anti-Rabbit IgG; 1:10,000 dilution

Tertiary Incubation

None

Detection

ECL picoplus/ECL femto

Results Summary

We treated HEK293T cells with 50% WNT3A-conditioned medium for 0, 0.5, 1, 2, 4, and 8 h, then separated cytosolic and nuclear fractions by hypotonic swelling, needle shearing, and differential centrifugation. Cytosolic fractions (30 µg) and equal volumes of the corresponding nuclear fractions, representing approximately equal cell equivalents, were resolved on 4–12% Bis-Tris gels and transferred to nitrocellulose. Membranes were blocked in 5% milk/TBS-T and probed with the LEF1 antibody at 1:1000 overnight at 4 °C, followed by HRP-conjugated donkey anti-rabbit IgG (1:10,000) and ECL pico plus/ECL femto. The antibody detected a doublet at approximately 50–55 kDa, exclusively in the nuclear fractions, with no detectable signal in any cytoplasmic lane. α-Tubulin blotted on the same membrane was confined to the cytoplasmic fractions, confirming that the fractions were well separated and that the nuclear LEF1 signal was not due to cytoplasmic carryover. We did not include a LEF1 knockout or knockdown control, so band identity was not confirmed genetically.

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Summary

The Good

The antibody is exceptionally clean. Across twelve lanes there was no background and no non-specific banding, and the nuclear-only signal was completely absent from cytoplasmic lanes, which is a strong indicator of specificity in a fractionation experiment. Detection of endogenous LEF1 in 293T cells worked at the recommended 1:1000 dilution in milk-based buffer. The antibody also resolves a LEF1 doublet, which is useful for anyone examining LEF1 isoforms or modified forms.

The Bad

None

The Bottom Line

CST's LEF1 antibody gives a clean, nuclear-restricted signal for endogenous LEF1 and performed excellently in subcellular fractionation. I recommend it, with the standard caveat that we did not confirm specificity with a LEF1-null control.

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