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CACNA2D1 / Voltage-dependent calcium channel subunit alpha-2/delta-1 · Western blot design guide
Design a Western Blot for CACNA2D1
Source-linked CACNA2D1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CACNA2D1 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
CACNA2D1 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 | ~124.6 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Skeletal muscle (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (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 | 5 isoform(s) |
Section 1
Source-Linked CACNA2D1 Western Blot Protocol Options
The A04282-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysate from NIH/3T3 cells, (catalog A04282-2) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A04282-2; use the WB datasheet starting dilution (standard starting point) |
| 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 CACNA2D1 Western Blot Band Size?
The predicted precursor is 124.6 kDa; glycosylation, cleavage, disulfide-linked chains and isoforms may affect migration, but no empirical band size is available.
| Band near 124.6 kDa | Compatible with the predicted precursor mass; band identity needs confirmation |
| Band above 124.6 kDa | N-linked glycosylation may increase apparent size |
| Band below 124.6 kDa | Signal-peptide removal or separation of the alpha-2 and delta-1 chains may contribute |
| Different bands under reducing and non-reducing conditions | The disulfide links the alpha-2 and delta-1 chains; it does not indicate a homodimer |
| Multiple bands | Isoforms 1–5 or processing may contribute, but distinct migration is unconfirmed |
💡Expected CACNA2D1 appearanceUniProt predicts a 124.6 kDa precursor; signal-peptide cleavage, N-linked glycosylation, linked alpha-2/delta-1 chains and isoforms may affect migration, but no empirical band size is supplied, so confirm identity with appropriate controls.
| UniProt precursor mass | Predicts 124.6 kDa before accounting for processing or modifications |
| N-linked glycosylation at Asn92, Asn136, Asn184, Asn324, Asn348, Asn468, Asn475 and Asn604 | May raise apparent size; the listed sites do not establish a shift or smear |
| Signal peptide at residues 1–24 | Removal makes the mature chain smaller than the precursor |
| Disulfide between residues 404 and 1059 | Links alpha-2 and delta-1 chains under non-reducing conditions; reduction may separate them |
| Splice isoforms 1, 2, 3, 4 and 5 | May differ in size; their masses and migration differences are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane protein may be poorly recovered or solubilized | Check membrane extraction and include a positive-control sample |
| Band higher than expected | N-linked glycosylation may affect migration | Compare with a deglycosylated aliquot and verify band identity |
| Band lower than expected | Signal-peptide removal or separation of alpha-2 and delta-1 chains may contribute | Compare reducing and non-reducing lanes and verify antibody target recognition |
| Broad smear instead of sharp band | Variation in N-linked glycosylation is possible | Compare with a deglycosylated aliquot and assess sample integrity |
| Multiple bands | Isoforms or processing may contribute; their migration is unconfirmed | Compare reducing conditions and confirm bands with an independent antibody |
| Weak or no signal | Membrane extraction may limit target recovery | Check extraction efficiency and include a positive-control lysate |
Experiment evidence
Western blot images showing CACNA2D1 band
A04282-2 · CACNA2D1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CACNA2D1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CACNA2D1 in Western blot, you can use skeletal muscle lysate.
Positive control: Skeletal muscle (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside samples.
⚠️Feasibility: As a membrane protein, CACNA2D1 may be easier to detect in membrane-enriched lysates.
HPA tissue expression evidence for CACNA2D1
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 |
|---|---|---|---|---|
| Skeletal muscle | myocytes | High | Protein (IHC) | HPA → |
| Colon | peripheral nerve/ganglion | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Medium | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| 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 → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CACNA2D1 Western Blot Tips
Deeper troubleshooting and optimisation questions for CACNA2D1, answered from its protein features.
How should CACNA2D1 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.
How could CACNA2D1 isoforms affect band interpretation?
Isoforms · UniProt lists five isoforms. In canonical UniProt numbering, isoforms 3 and 4 lack residues 531–554; isoforms 2 and 5 lack 531–549; and isoforms 2 and 3 replace Y644 with SKKGKMKD. Check whether the antibody epitope overlaps these regions before attributing different bands to isoforms.
Which CACNA2D1 glycosylation sites matter when assessing a band?
PTM · UniProt lists 16 N-linked sites at canonical positions 92, 136, 184, 324, 348, 468, 475, 604, 613, 675, 781, 824, 888, 895, 985, and 998. Consider glycosylation when interpreting migration, while treating any proposed band shift as unconfirmed without experimental evidence.
Yes. UniProt lists phosphoserine at position 119 in canonical numbering. That annotation alone does not establish when phosphorylation occurs or whether it produces a resolvable Western blot shift.
Yes. UniProt lists phosphoserine at position 119 in canonical numbering. That annotation alone does not establish when phosphorylation occurs or whether it produces a resolvable Western blot shift.
Does this guide establish induction of CACNA2D1?
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 CACNA2D1 Western blot?
Transfer · CACNA2D1 is listed as a single-pass membrane protein with a predicted mass of 124.6 kDa. Check that the band region transfers efficiently by examining the gel and membrane after transfer. The supplied features do not specify one transfer method or setting.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04282-2 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 CACNA2D1 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.
Why might CACNA2D1 migrate away from its predicted 124.6 kDa?
Interpretation · The 124.6 kDa value is predicted from sequence. UniProt lists a signal peptide at residues 1–24, 16 N-linked glycosylation sites, and five isoforms. These features are relevant when assessing apparent mass, but the supplied record gives no observed band size and does not establish a visible shift.
Use the same transfer and detection conditions across samples, and quantify a consistently defined band region. Because UniProt lists five isoforms, 16 N-linked sites, and disulfide-linked alpha-2-1 and delta-1 chains, verify that the bands being compared represent the same species.
Check antibody epitope coverage against the listed isoform changes at canonical positions 531–554 and 644. Also consider the 16 N-linked sites and the disulfide-linked alpha-2-1/delta-1 chains when comparing reducing and nonreducing samples. These features suggest possibilities; they do not identify an unexpected band on their own.
Use the same transfer and detection conditions across samples, and quantify a consistently defined band region. Because UniProt lists five isoforms, 16 N-linked sites, and disulfide-linked alpha-2-1 and delta-1 chains, verify that the bands being compared represent the same species.
Check antibody epitope coverage against the listed isoform changes at canonical positions 531–554 and 644. Also consider the 16 N-linked sites and the disulfide-linked alpha-2-1/delta-1 chains when comparing reducing and nonreducing samples. These features suggest possibilities; they do not identify an unexpected band on their own.
Boster reagents
CACNA2D1 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-Cacna2d1 Antibody
Cat # A04282-2
The catalog reports anti-CACNA2D1 antibody A04282-2 for Western blot, with stated human, mouse, and rat reactivity. Its WB image uses NIH/3T3 cell lysate; the supplied evidence does not show WB results in human or rat samples.
Which to pick: A04282-2 is the only listed option. Its WB image documents NIH/3T3 cell lysate, so use that example when assessing fit for your sample; human and rat reactivity is listed without corresponding WB examples here.
Source: BosterBio CACNA2D1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P54289.
- Human Protein Atlas. CACNA2D1 tissue expression.
- PMC6208517 — target-verified WB comparison
- PMC10940998 — target-verified WB comparison
- PMC13508336 — target-verified WB comparison