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- Table of Contents
ABCD3 / ATP-binding cassette sub-family D member 3 · Western blot design guide
Design a Western Blot for ABCD3
Source-linked ABCD3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ABCD3 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
ABCD3 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 | ~75.5 kDa | |
| Observed band | ~70 kDa | |
| Gel | 10% (catalog PA2091) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Ovary (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 Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked ABCD3 Western Blot Protocol Options
The PA2091 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human HepG2, rat liver, mouse liver (catalog PA2091) |
| Gel % | 10% (catalog PA2091) |
| Load | 30 ug; reducing conditions (catalog PA2091) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA2091) |
| Membrane | nitrocellulose membrane (catalog PA2091) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA2091) |
| Primary antibody | PA2091 · 0.5 μg/mL (catalog PA2091) |
| Primary incubation | overnight at 4°C (catalog PA2091) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA2091) |
| Secondary incubation | 1.5 hour at RT (catalog PA2091) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA2091) |
| Detection | ECL (catalog PA2091) |
Section 2
What Is the Expected ABCD3 Western Blot Band Size?
ABCD3 is predicted at 75.5 kDa and observed near 70 kDa; the cause of this difference is not established.
| Band near 70 kDa | Empirical ABCD3 band observed in reducing whole-cell and tissue lysates |
| Band near 75.5 kDa | Near the UniProt predicted mass; identity requires confirmation |
| Higher band near twice the monomer size | Could reflect an ABCD3 homodimer if it survives sample preparation |
| Additional bands at different sizes | Could reflect isoforms 1, 2, and 3, whose migration differences are unknown |
💡Expected ABCD3 appearanceUniProt predicts 75.5 kDa, while antibody QC shows an approximately 70 kDa band in reducing lysates; confirm band identity with an independent antibody or ABCD3 depletion.
| UniProt predicted mass | 75.5 kDa for the reference sequence; the empirical band is approximately 70 kDa |
| N-linked glycosylation at Asn12 | Potential glycan addition could alter migration; a visible shift is not established |
| N-linked glycosylation at Asn106 | Potential glycan addition could alter migration; a visible shift is not established |
| N-linked glycosylation at Asn206 | Potential glycan addition could alter migration; a visible shift is not established |
| ABCD3 homodimer formation | A surviving dimer could migrate near twice the monomer size |
| Isoforms 1, 2, and 3 | Their sizes may differ, but distinct band positions are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ABCD3 is a peroxisomal membrane protein that may extract poorly | Check membrane solubilization and enrich the membrane fraction |
| Band higher than expected | A homodimer may persist through sample preparation | Compare reducing and more strongly denaturing conditions |
| Band lower than expected | The observed 70 kDa band runs below the 75.5 kDa prediction; the cause is unknown | Confirm identity by ABCD3 depletion or an independent antibody |
| Broad smear instead of sharp band | Variable modification at the listed N-linked glycosylation sites is possible but unproven | Compare treated and untreated samples with a suitable N-glycan removal control |
| Multiple bands | Isoforms 1, 2, and 3 are listed, but their band positions are unknown | Test band identity with ABCD3 depletion and an independent antibody |
Experiment evidence
Western blot images showing ABCD3 band
PA2091 · ABCD3
M02370 · ABCD3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ABCD3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ABCD3 in Western blot, you can use adrenal gland lysate, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Ovary (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As ABCD3 is a multi-pass peroxisomal membrane protein, efficient membrane protein extraction may be needed.
HPA tissue expression evidence for ABCD3
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | granular cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ABCD3 Western Blot Tips
Deeper troubleshooting and optimisation questions for ABCD3, answered from its protein features.
How should ABCD3 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 ABCD3 isoforms produce additional bands?
Isoforms · Three isoforms are listed. Relative to the canonical sequence, isoform 2 lacks residues 277–386; isoform 3 replaces residues 229–236 and lacks 237–659. These changes could alter apparent size, but the features do not establish which isoforms appear on a blot.
Could glycosylation change ABCD3 migration?
PTM · UniProt lists N-linked glycosylation at Asn12, Asn106 and Asn206, using canonical sequence coordinates. A glycosylation-dependent shift is possible, but site annotations alone do not show that any particular band is shifted.
UniProt lists N6-acetyllysine at positions 61, 260, 399 and 533, and phosphoserine at 659. These are canonical sequence coordinates. Their presence does not demonstrate a visible shift or explain the approximately 70 kDa band.
UniProt lists N6-acetyllysine at positions 61, 260, 399 and 533, and phosphoserine at 659. These are canonical sequence coordinates. Their presence does not demonstrate a visible shift or explain the approximately 70 kDa band.
Does this guide establish induction of ABCD3?
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 ABCD3 Western blot?
Transfer · ABCD3 is a multi-pass peroxisomal membrane protein with a predicted mass of 75.5 kDa. Check both the post-transfer gel and membrane when optimizing transfer. The supplied features do not identify a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the PA2091 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 ABCD3 bands be quantified?
Quantitation · Define the band being measured and use the same band across samples. ABCD3 has three listed isoforms, so combining bands could mix distinct forms. The features alone cannot establish which band corresponds to each isoform.
Why might ABCD3 run near 70 kDa instead of 75.5 kDa?
Interpretation · The canonical predicted mass is 75.5 kDa, and the reported apparent band is about 70 kDa. The supplied features do not establish why they differ. Check band identity before assigning the difference to a modification or isoform.
ABCD3 forms homodimers and can heterodimerize with ABCD1 or ABCD2. A higher band could prompt investigation of these complexes, but oligomerization alone does not identify an unexpected band or show that a dimer survives blot preparation.
ABCD3 forms homodimers and can heterodimerize with ABCD1 or ABCD2. A higher band could prompt investigation of these complexes, but oligomerization alone does not identify an unexpected band or show that a dimer survives blot preparation.
Boster reagents
ABCD3 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-PMP70/ABCD3 Antibody Picoband®
Cat # PA2091
Real WB data
Anti-PMP70 ABCD3 Monoclonal Antibody
Cat # M02370
PA2091 and M02370 both list human, mouse, and rat reactivity and have WB images. PA2091 documents human cell and rat and mouse liver lysates with a band near 70 kDa. M02370’s caption identifies only HepG2 lysate.
Which to pick: Choose PA2091 for documented WB examples spanning the listed species and a reported primary antibody concentration of 0.5 μg/mL. M02370 has a HepG2 WB image, but its caption gives few experimental details.
Source: BosterBio ABCD3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P28288.
- Human Protein Atlas. ABCD3 tissue expression.
- PMC8647813 — target-verified WB comparison
- PMC6588346 — target-verified WB comparison
- PMC8578467 — target-verified WB comparison