CDX2 / Homeobox protein CDX-2 · Western blot design guide

Design a Western Blot for CDX2

Real validated CDX2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CDX2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.

Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use

Western blot protocol sheet for CDX2: expected band ~33.5 kDa, hero antibody A00877-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **CDX2** Western blot protocol sheet — expected band ~33.5 kDa, antibody A00877-1, controls and PMC citations. Open the full CDX2 WB guide →

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CDX2 Western Blot Experimental Design Guide

Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before running

	Expected band	~33.5 kDa
	Gel	12–15% (standard starting point)
	Positive control ⓘ	Appendix (IHC candidate; verify WB) +4 more
	Negative control ⓘ	Adipose tissue (IHC candidate; verify WB)

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated + Ubl conjugation
	Caveat	Modification-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	1 isoform(s)

Section 1

Real Curated CDX2 Western Blot Protocols

The A00877-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	mouse brain tissue lysate (catalog A00877-1)
Gel %	12–15% (standard starting point)
Load	20–30 µg total protein per lane; optimize for abundance (standard starting point)
Transfer	Standard semi-dry transfer; verify efficiency (standard starting point)
Membrane	0.45 µm PVDF (standard starting point)
Blocking	5% milk or 5% BSA in TBST (standard starting point)
Primary antibody	A00877-1 · 1 μg/mL (catalog A00877-1)
Primary incubation	Overnight at 4 °C (standard starting point)
Secondary antibody	Species-matched HRP conjugate at validated dilution (standard starting point)
Secondary incubation	1 h at room temperature (standard starting point)
Wash	3 × 5 min in TBST (standard starting point)
Detection	ECL; bracket exposures to avoid saturation (standard starting point)

Section 2

What Is the Expected CDX2 Western Blot Band Size?

CDX2 has a predicted monomer mass of 33.5 kDa; phosphorylation and homodimerization are possible influences, but altered blot migration is not demonstrated.

What am I looking at on my blot?
Band near 33.5 kDa	Consistent with the predicted CDX2 monomer; confirm identity with controls.
Close doublet near 33.5 kDa	Phosphorylation at Ser60 or Ser283 is possible, but a mobility shift is unproven.
Band near 67 kDa	Could reflect a retained CDX2 homodimer; confirm identity because DNA-binding dimerization does not establish a stable blot band.
Little or no band in a cytoplasmic fraction	Consistent with CDX2 nuclear localization.

💡Expected CDX2 appearanceUniProt predicts a 33.5 kDa CDX2 monomer; no empirical band size or feature-dependent migration shift is established, so confirm a candidate band with ordinary identity controls.

How each factor affects band size
UniProt predicted mass	Sets the CDX2 monomer reference at 33.5 kDa.
Phosphoserine at Ser60	May affect mobility, but no visible shift is established.
Phosphoserine at Ser283	May affect mobility, but no visible shift is established.
CDX2 homodimerization	Could yield a band near twice the monomer size if the complex survives sample preparation; blot persistence is unproven.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear CDX2 may be poorly recovered during extraction.	Check nuclear extraction and a nuclear fraction.
Band higher than expected	A retained homodimer is possible but unverified on a denaturing blot.	Check sample denaturation and confirm the band with an independent antibody or CDX2 loss control.
Band lower than expected	The supplied features identify no cleavage that explains a smaller band.	Check sample integrity and confirm identity with a CDX2 loss control.
Multiple bands	Ser60 or Ser283 phosphorylation could affect mobility, but separate bands are unproven.	Compare phosphatase-treated and untreated samples and confirm CDX2 identity.
Weak or no signal	Nuclear CDX2 may be underrepresented in the tested fraction.	Test a nuclear extract and verify extraction with a nuclear marker.

Source: UniProt Q99626 Human Protein Atlas product datasheet (A00877-1)

Experiment evidence

Western blot images showing CDX2 band

A00877-1 · CDX2

M00877-1 · CDX2

A00877-3 · CDX2

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for CDX2 Western blot

🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CDX2 in Western blot, you can use appendix tissue, where HPA reports high expression.

Positive control: Appendix (IHC candidate; verify WB)

Negative control: Adipose tissue (IHC candidate; verify WB)

Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.

⚠️Feasibility: CDX2 is nuclear, so nuclear-enriched lysate may give a clearer signal.

HPA tissue expression evidence for CDX2

Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Higher expression tissues · candidate positive controls from IHC

Tissue	Cell type	Level	Evidence	Source
Appendix	endocrine cells	High	Protein (IHC)	HPA →
Colon	endocrine cells	High	Protein (IHC)	HPA →
Duodenum	endocrine cells	High	Protein (IHC)	HPA →
Rectum	endocrine cells	High	Protein (IHC)	HPA →
Small intestine	endocrine cells	Medium	Protein (IHC)	HPA →

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	adipocytes	Not detected	Protein (IHC)	HPA →
Bronchus	respiratory epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CDX2 Western Blot Tips

Deeper troubleshooting and optimisation questions for CDX2, answered from its protein features.

How should CDX2 band migration be interpreted?

Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.

Could CDX2 isoforms explain multiple bands?

Isoforms · The supplied features list one isoform and no alternative sequence. They do not support assigning multiple bands to CDX2 isoforms.

Which CDX2 phosphorylation sites matter when interpreting bands?

PTM · UniProt lists phosphoserine at positions 60 and 283. These are UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible mobility shift.

CDX2 has annotated phosphoserines at UniProt positions 60 and 283, but the supplied features do not establish an apparent band size or show that phosphorylation causes a visible shift.

Does this guide establish induction of CDX2?

Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.

How should transfer be checked for CDX2?

Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A00877-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.

How should CDX2 be quantified?

Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.

What size should CDX2 appear on a Western blot?

Interpretation · The predicted mass is 33.5 kDa. No observed band size is supplied, so use 33.5 kDa as a reference rather than an expected apparent size.

CDX2 is annotated as nuclear. For a nuclear fraction, compare samples prepared the same way and normalize to a suitable nuclear loading measure. The features provide no treatment or induction condition to assume.

CDX2 can bind DNA as a monomer or homodimer, but that annotation does not establish that a dimer survives Western blot preparation. Do not assign a higher band to a dimer from size alone.

Boster reagents

CDX2 Western Blot Antibodies

Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.

Real WB data Western blot analysis of CDX2 in mouse brain tissue lysate with CDX2 antibody at 1 μg/mL.

Anti-Homeobox protein CDX-2 CDX2 Antibody

Cat # A00877-1

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Real WB data

Western blot analysis of CDX2 using anti-CDX2 antibody (M00877-1). <br>
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. <br>
Lane 1: human 293T whole cell lysates,<br>
Lane 2: human Caco-2 whole cell lysates.<br>
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CDX2 antigen affinity purified monoclonal antibody (Catalog # M00877-1) at 1:1000 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:500 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CDX2 at approximately 37 kDa. The expected band size for CDX2 is at 34 kDa.

Anti-CDX2 Rabbit Monoclonal Antibody

Cat # M00877-1

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Real WB data

Western blot analysis of CDX2 using anti-CDX2 antibody (A00877-3). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human SW620 whole cell lysates, Lane 2: human CACO-2 whole cell lysates, Lane 3: human DLD1 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CDX2 antigen affinity purified polyclonal antibody (Catalog # A00877-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CDX2 at approximately 45 kDa. The expected band size for CDX2 is at 33 kDa.

Anti-CDX2 Antibody Picoband®

Cat # A00877-3

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Three the supplier anti-CDX2 antibodies have WB images: A00877-1 in mouse brain lysate, M00877-1 in human 293T and Caco-2 lysates, and A00877-3 in human SW620, CACO-2 and DLD1 lysates. The latter two report bands above their expected sizes.

Which to pick: For human lysates, compare M00877-1 (reported 37 kDa; expected 34 kDa) with A00877-3 (reported 45 kDa; expected 33 kDa). For mouse brain lysate, A00877-1 has the matching image. A00877-1 and M00877-1 list human, mouse and rat reactivity; A00877-3 lists human.

Source: BosterBio CDX2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry Q99626.
Human Protein Atlas. CDX2 tissue expression.
PMC6158822 — target-verified WB comparison
PMC7993706 — target-verified WB comparison
PMC7494815 — target-verified WB comparison