Colorectal Lineage Markers by Western Blot: CDX2, SATB2, CK20, CDH17, Villin & MUC2

A colorectal lineage marker panel answers a question that proliferation or apoptosis markers cannot: not whether a tissue is malignant, but where it came from. When you need to confirm that a cell line, xenograft, organoid, or tumor lysate carries a colorectal epithelial program — or to track loss of that program during progression — six intestinal lineage and differentiation markers read together (CDX2, SATB2, cytokeratin 20, CDH17, villin, and MUC2) are far more informative than any single general marker. This application note describes six U-Blot® monoclonal antibodies for studying intestinal identity in colorectal cancer (CRC) models by Western blot (WB), with the rationale for each marker, how to read them as a system, and the interpretive caveats that keep conclusions defensible. All six are validated for WB and immunofluorescence, and most for IHC and ELISA as well.

Two layers of “colorectal specificity”

No protein is expressed exclusively in colorectal tumors, so “colorectal specificity” comes from two complementary ideas. The first is tissue of origin: transcription factors and structural proteins that mark cells as intestinal-lineage (CDX2, SATB2, CK20, CDH17, villin, MUC2). The second is differentiation state: several of these same markers are progressively lost as tumors dedifferentiate, so their reduction or absence carries biological and, in some settings, prognostic meaning.

The panel is internally connected. CDX2 is a master regulator of intestinal differentiation that transcriptionally drives downstream intestinal genes, including MUC2 and CDH17.^1,8,9 These markers are therefore not six independent readouts but a partially hierarchical program — useful framing when interpreting why loss of an upstream regulator (CDX2) may coincide with attenuation of its targets.

The colorectal lineage marker panel at a glance

All six are UcallM Biosciences U-Blot® unconjugated monoclonals supplied at 50 µL and 100 µL. Recommended WB starting dilutions per datasheet: CDX2 1:1000; SATB2 1:2000–1:10000; CK20 1:500–1:2000; CDH17 1:2000–1:10000; villin 1:2000–1:10000; MUC2 1:2000–1:10000. Optimize per lysate.

×

Marker-by-marker rationale: CDX2, SATB2, CK20, CDH17, villin, MUC2

CDX2 (caudal-type homeobox 2) — a nuclear transcription factor and the central regulator of intestinal differentiation, observed at ~34 kDa. Beyond marking intestinal identity, loss of CDX2 defines a poorly differentiated, higher-risk subset: in a large study of stage II and III colon cancer, CDX2-negative tumors had significantly lower disease-free survival, and the marker has been proposed to aggregate several adverse features into a single readout.¹ CDX2 loss has also been linked to invasive behavior and tumor budding.² On WB, the CDX2 antibody is well suited to comparing expression across cell lines or documenting its loss in dedifferentiated models.

SATB2 (special AT-rich sequence-binding protein 2) — a nuclear-matrix DNA-binding protein with the most colorectal-restricted expression pattern of the group. It is positive in roughly 85% of CRCs, and SATB2 and/or CK20 together identify ~97% of colorectal carcinomas, making the pair a workhorse for establishing colorectal origin.^3,4 SATB2 loss has been associated with mismatch-repair deficiency and BRAF mutation, tying it to defined molecular subgroups.⁵ The SATB2 antibody runs higher than its calculated mass (~100 kDa observed vs. 83 kDa calculated).

Cytokeratin 20 (CK20) — the classic intestinal keratin, read as part of the CK20-positive / CK7-negative profile that characterizes colorectal adenocarcinoma. This clone shows no cross-reactivity with CK1, 5, 6, 7, 8, 13, 14, 15, 17, or 18. One interpretive caution worth building into any CK20 experiment: CK20 can be reduced in high-microsatellite-instability (MSI-H) CRC, so weak or absent CK20 does not by itself exclude colorectal origin.^6,7 Clean single band at ~48 kDa.

CDH17 (cadherin-17 / LI-cadherin) — an intestine-restricted membrane glycoprotein and a CDX2 transcriptional target. Among lineage markers it is the most sensitive for digestive-tract adenocarcinomas, positive in ~96–99% of colorectal cases and exceeding CDX2 and CK20 in sensitivity, though with somewhat lower specificity.^8,9 A large tissue-microarray survey further linked reduced CDH17 within CRC to higher pT stage, nodal metastasis, and MSI.¹⁰ Because it is glycosylated, observed MW (~120 kDa) exceeds the calculated 92 kDa.

Villin — a calcium-regulated actin-bundling protein of the enterocyte brush border, and a marker of intestinal/epithelial differentiation frequently used alongside CK7/CK20 to assign adenocarcinoma origin. The villin antibody is best treated as a differentiation/lineage marker rather than a prognostic one: in a whole-slide stage II colon cancer study, villin (unlike MUC2) showed no significant independent association with outcome.¹¹ Observed at ~93 kDa.

MUC2 (intestinal mucin-2) — the major secreted gel-forming mucin of the colon and a marker of goblet-cell / mucinous differentiation. Loss of MUC2 has been associated with reduced survival in stage II colon cancer and in TCGA expression data,¹¹ while the broader prognostic picture of mucinous differentiation is nuanced and context-dependent.¹² MUC2 is the panel’s most technically demanding blot: an enormous, heavily O-glycosylated, multimerizing glycoprotein (calculated ~551 kDa) that does not migrate at its calculated mass; this clone reports an observed band of ~140–170 kDa.

×

Reading the panel as a system

The markers are most powerful in combination rather than individually. For establishing colorectal identity, the CK20-positive/CK7-negative pattern plus SATB2 covers the large majority of cases, CDH17 adds sensitivity where CDX2 or CK20 are weak, and CDX2 and MUC2 layer on differentiation information. Because CDX2 sits upstream of MUC2 and CDH17, coordinated loss of an upstream regulator and its targets is biologically coherent and can be tracked together across a model series. For dedifferentiation or progression studies, the informative signal is often the reduction of CDX2, SATB2, CK20, or MUC2 rather than their presence.

Western blot practical notes for the lineage-marker panel

Expect non-calculated migration. SATB2 (~100 vs. 83 kDa), CDH17 (~120 vs. 92 kDa, glycosylation), and especially MUC2 (~140–170 kDa vs. 551 kDa calculated) run away from their theoretical masses; treat the observed values in the table above as the expected bands rather than rejecting them as artifacts. For MUC2, use low-percentage gels, allow extended/optimized transfer for the high-MW species, and be aware of the smeared or laddered signal typical of mucins.

Match species to model. SATB2, CK20, and MUC2 are validated for human, mouse, and rat; CDX2 (human, monkey), CDH17 (human), and villin (human) are not validated on rodent samples. For mouse or rat colon models, build the core panel from SATB2, CK20, and MUC2 and validate the others in-house before use.

Controls. Include a colorectal-positive lysate and a lineage-negative lysate on every blot, plus a housekeeping loading control. Well-characterized human colon adenocarcinoma lines are common positive controls (for example, differentiated enterocyte-like lines for CDX2/villin and goblet-like lines for MUC2); confirm marker status for the specific line and passage in your own hands, since expression drifts with differentiation state.

×

Interpretation caveats

Featured products

Browse Antibodies Request Bulk Pricing

Frequently asked questions

References

1. Dalerba P, Sahoo D, Paik S, et al. CDX2 as a prognostic biomarker in stage II and stage III colon cancer. N Engl J Med. 2016;374(3):211–222. PubMed
2. CDX2 loss in colorectal cancer cells is associated with invasive properties and tumor budding. Sci Rep. 2025;15. DOI
3. Magnusson K, de Wit M, Brennan DJ, et al. SATB2 in combination with cytokeratin 20 identifies over 95% of all colorectal carcinomas. Am J Surg Pathol. 2011;35(7):937–948. PubMed
4. Dragomir A, de Wit M, Johansson C, et al. The role of SATB2 as a diagnostic marker for tumors of colorectal origin. Am J Clin Pathol. 2014;141(5):630–638. PubMed
5. Ma C, Olevian DC, Lowenthal BM, et al. Loss of SATB2 expression in colorectal carcinoma is associated with DNA mismatch repair protein deficiency and BRAF mutation. Am J Surg Pathol. 2018;42(10):1409–1417. PubMed
6. McGregor DK, Wu TT, Rashid A, Luthra R, Hamilton SR. Reduced expression of cytokeratin 20 in colorectal carcinomas with high levels of microsatellite instability. Am J Surg Pathol. 2004;28(6):712–718. PubMed
7. Bayrak R, Haltas H, Yenidunya S. The value of CDX2 and cytokeratins 7 and 20 expression in differentiating colorectal adenocarcinomas from extraintestinal GI adenocarcinomas. Diagn Pathol. 2012;7:9. PubMed
8. Su MC, Yuan RH, Lin CY, Jeng YM. Cadherin-17 is a useful diagnostic marker for adenocarcinomas of the digestive system. Mod Pathol. 2008;21(11):1379–1386. PubMed
9. Panarelli NC, Yantiss RK, Yeh MM, Liu Y, Chen YT. Tissue-specific cadherin CDH17 is a useful marker of gastrointestinal adenocarcinomas with higher sensitivity than CDX2. Am J Clin Pathol. 2012;138(2):211–222. PubMed
10. Jacobsen F, Pushpadevan R, Viehweger F, et al. Cadherin-17 (CDH17) expression in human cancer: a tissue microarray study on 18,131 tumors. Pathol Res Pract. 2024;256:155175. PubMed
11. Cecchini MJ, Walsh JC, Parfitt J, et al. CDX2 and Muc2 immunohistochemistry as prognostic markers in stage II colon cancer. Hum Pathol. 2019;90:70–79. PubMed
12. Elomaa H, Tarkiainen V, Äijälä VK, et al. Associations of mucinous differentiation and mucin expression with immune cell infiltration and prognosis in colorectal adenocarcinoma. Br J Cancer. 2025. DOI