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- Table of Contents
CNTNAP2 / Contactin-associated protein-like 2 · Western blot design guide
Design a Western Blot for CNTNAP2
Source-linked CNTNAP2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CNTNAP2 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
CNTNAP2 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~148.2 kDa | |
| Observed band | ~170 kDa | |
| Gel | 5–20% (catalog A02819-2) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +3 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked CNTNAP2 Western Blot Protocol Options
The A02819-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat brain, mouse brain (catalog A02819-2) |
| Gel % | 5–20% (catalog A02819-2) |
| Load | 30 ug; reducing conditions (catalog A02819-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02819-2) |
| Membrane | nitrocellulose membrane (catalog A02819-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02819-2) |
| Primary antibody | A02819-2 · 0.5 μg/mL (catalog A02819-2) |
| Primary incubation | overnight at 4°C (catalog A02819-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02819-2) |
| Secondary incubation | 1.5 hour at RT (catalog A02819-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02819-2) |
| Detection | ECL (catalog A02819-2) |
Section 2
What Is the Expected CNTNAP2 Western Blot Band Size?
CNTNAP2 is predicted at 148.2 kDa and observed near 170 kDa in reducing brain lysates; the cause of the difference is not established.
| Band near 170 kDa | Observed CNTNAP2 band in reducing brain lysates; identity still requires antibody controls |
| Band near the predicted 148.2 kDa | Could reflect the predicted precursor mass; migration at this position is not established |
| Band slightly below a precursor band | Could reflect cleavage of the 27-residue signal peptide |
| Broader or higher band | Could reflect N-linked glycosylation; its visible effect is unverified |
| Multiple bands of different sizes | Could reflect isoforms 1 and 2; distinct migration is unverified |
💡Expected CNTNAP2 appearanceCNTNAP2 has a predicted precursor mass of 148.2 kDa, while a ~170 kDa band was observed in reducing brain lysates; confirm band identity with antibody controls because the difference is unexplained.
| Predicted precursor mass | 148.2 kDa provides a sequence-based reference, while the observed band is ~170 kDa |
| N-linked glycosylation at Asn289, Asn346, Asn363, Asn379, Asn436, Asn506, Asn507, and Asn546 | Could increase apparent size or broaden a band; the contribution is unmeasured |
| Signal peptide at residues 1–27 | Cleavage makes the mature protein slightly smaller than its precursor; the migration effect is unmeasured |
| Isoforms 1 and 2 | May differ in size, but their relative masses and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated CNTNAP2 may be poorly recovered | Check membrane solubilization and use brain lysate as a positive control |
| Band higher than expected | N-linked glycosylation may alter migration; the cause of the ~170 kDa band is unestablished | Compare matched samples with and without N-glycan removal and confirm antibody specificity |
| Band lower than expected | Signal-peptide cleavage or protein fragmentation may contribute; signal cleavage alone does not establish a large shift | Check the antibody epitope and compare fresh lysate with the original sample |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation is possible | Compare matched samples with and without N-glycan removal |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct bands are not established | Check antibody specificity and compare samples with different CNTNAP2 expression |
| Fragments below expected size | CNTNAP2 may have degraded during sample handling | Prepare fresh lysate with protease inhibitors and check whether the smaller bands persist |
Experiment evidence
Western blot images showing CNTNAP2 band
A02819-2 · CNTNAP2
M02819 · CNTNAP2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CNTNAP2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CNTNAP2 in Western blot, you can use cerebellum lysate, which has high HPA expression.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As an axonal membrane protein, CNTNAP2 may require effective membrane protein extraction for a clear signal.
HPA tissue expression evidence for CNTNAP2
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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CNTNAP2 Western Blot Tips
Deeper troubleshooting and optimisation questions for CNTNAP2, answered from its protein features.
How should CNTNAP2 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 CNTNAP2 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 1–1223 of the canonical sequence, so it could differ substantially in size and antibody recognition. Check whether the antibody epitope is present in each isoform before assigning a band; the sequence annotation does not establish that both isoforms are expressed or detectable in your sample.
Which glycosylation sites matter when interpreting CNTNAP2 bands?
PTM · The annotated N-linked sites are at canonical UniProt positions 289, 346, 363, 379, 436, 506, 507, 546, 630, 735, 1116 and 1198. Their presence makes glycosylation relevant to band interpretation, but does not establish site occupancy or a visible shift. Keep UniProt numbering explicit when comparing these positions with antibody or paper numbering.
UniProt annotates phosphoserines at canonical positions 1303 and 1306. These sites establish that phosphorylation is reported, but do not show that it causes a visible shift or explains the roughly 22 kDa observed-versus-predicted difference. If testing phosphorylation, compare matched samples and identify the band before interpreting a mobility change.
UniProt annotates phosphoserines at canonical positions 1303 and 1306. These sites establish that phosphorylation is reported, but do not show that it causes a visible shift or explains the roughly 22 kDa observed-versus-predicted difference. If testing phosphorylation, compare matched samples and identify the band before interpreting a mobility change.
Does this guide establish induction of CNTNAP2?
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.
What transfer method to use for CNTNAP2 Western blot?
Transfer · CNTNAP2 is a single-pass membrane protein with a predicted mass of 148.2 kDa and an observed band near 170 kDa. Choose and verify a transfer setup that recovers proteins in that size range; inspect transfer near 170 kDa before interpreting a missing band. The supplied features do not establish one transfer method as best.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02819-2 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 CNTNAP2 bands be quantified?
Quantitation · Quantify the same identified CNTNAP2 band across samples, using the approximately 170 kDa observed band as a starting reference. Record sample source consistently: CNTNAP2 is annotated at membranes, axons and paranodal junctions, so differing sample composition could affect the measured signal. Keep bands attributed to distinct isoforms separate unless their identities are established.
Why might CNTNAP2 run near 170 kDa instead of 148.2 kDa?
Interpretation · The observed band is about 170 kDa, while the predicted mass is 148.2 kDa. CNTNAP2 has 12 annotated N-linked glycosylation sites and a 1–27 signal peptide, but these features alone do not establish the cause or size of the difference. Use the observed band as a reference and confirm its identity before assigning it to CNTNAP2.
Check the antibody epitope against the isoform 2 deletion of canonical residues 1–1223. A lower band cannot be assigned to isoform 2 from size alone. The 1–27 signal peptide and annotated modifications also do not establish the identity of an unexpected band; confirm it with evidence specific to the band and sample.
Check the antibody epitope against the isoform 2 deletion of canonical residues 1–1223. A lower band cannot be assigned to isoform 2 from size alone. The 1–27 signal peptide and annotated modifications also do not establish the identity of an unexpected band; confirm it with evidence specific to the band and sample.
Boster reagents
CNTNAP2 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
Anti-Caspr2/CNTNAP2 Antibody Picoband®
Cat # A02819-2
Real WB data
Anti-CNTP2 Rabbit Monoclonal Antibody
Cat # M02819
Two the supplier anti-CNTNAP2 antibodies list Human, Mouse, and Rat reactivity and have WB images. A02819-2 shows an approximately 170 kDa band in rat and mouse brain lysates; M02819 shows mouse brain lysate. The supplied captions do not document a human WB sample.
Which to pick: Choose A02819-2 if you want documented rat and mouse brain WB conditions, including a 0.5 μg/mL primary concentration. M02819 is the listed rabbit monoclonal option with a mouse brain WB image, but its caption provides fewer experimental details.
Source: BosterBio CNTNAP2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UHC6.
- Human Protein Atlas. CNTNAP2 tissue expression.
- PMC11100810 — target-verified WB comparison
- PMC10904759 — target-verified WB comparison
- PMC13461343 — target-verified WB comparison