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- Table of Contents
EHHADH / Peroxisomal bifunctional enzyme · Western blot design guide
Design a Western Blot for EHHADH
Real validated EHHADH Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EHHADH 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
EHHADH 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 | ~79.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Liver (IHC candidate; verify WB) +1 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated EHHADH Western Blot Protocols
The A05757-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Jurkat, Raw264.7, H9C2 (catalog A05757-2) |
| 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 | A05757-2; 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 EHHADH Western Blot Band Size?
EHHADH has a predicted monomer mass of 79.5 kDa; splice isoforms could affect migration, but no empirical band size or distinct isoform bands are established.
| Band near 79.5 kDa | consistent with the predicted monomer, pending identity controls |
| Additional discrete band of unspecified size | could reflect isoform 1 or 2; their migration is uncharacterized |
| Single band despite two annotated isoforms | the isoforms may not resolve as separate bands |
| Faint band in a cytosolic fraction | consistent with peroxisomal localization |
💡Expected EHHADH appearanceUniProt predicts a 79.5 kDa monomer, but no empirical band size is supplied; confirm any band near that size with antibody and EHHADH depletion controls.
| Predicted monomer mass | places the reference band near 79.5 kDa |
| Splice isoform 1 | its individual apparent mass is not supplied |
| Splice isoform 2 | its individual apparent mass and size relative to isoform 1 are not supplied |
| Alternative splicing of isoforms 1 and 2 | may affect migration, but distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | peroxisomal protein may be poorly recovered during extraction | check lysate recovery with a peroxisomal marker and an EHHADH positive control |
| Band higher than expected | identity or migration of the band is unverified | compare with an EHHADH depletion control |
| Band lower than expected | isoform migration or protein degradation is possible but unestablished | compare fresh lysate with an EHHADH depletion control |
| Multiple bands | two splice isoforms are annotated, but separate migration is unproven | check which bands disappear after EHHADH depletion |
| Weak or no signal | EHHADH abundance or peroxisomal recovery may be low in the sample | check loading, peroxisomal recovery, and an EHHADH positive control |
Experiment evidence
Western blot images showing EHHADH band
A05757-2 · EHHADH
A05757-3 · EHHADH
A05757-3 · EHHADH
A05757-1 · EHHADH
A05757-1 · EHHADH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EHHADH Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EHHADH in Western blot, you can use liver lysate, which has high HPA expression.
Positive control: Liver (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: Liver offers a strong positive, while HPA reports no detectable expression in adipose tissue for a negative control.
HPA tissue expression evidence for EHHADH
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 |
|---|---|---|---|---|
| Liver | hepatocytes | High | Protein (IHC) | HPA → |
| Kidney | proximal tubules (cell body) | 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 EHHADH Western Blot Tips
Deeper troubleshooting and optimisation questions for EHHADH, answered from its protein features.
What should I check if unexpected EHHADH bands appear?
Band shift · Compare band positions with the 79.5-kDa canonical prediction and the shorter isoform 2. Check antibody epitope coverage and whether bands vary with sample preparation. The supplied features list no signal peptide, propeptide or glycosylation sites; the listed modifications alone cannot explain an observed mass difference.
How can the two EHHADH isoforms affect band interpretation?
Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks canonical residues 1–96. A lower band could be consistent with isoform 2, but band position alone cannot identify it. Check whether the antibody epitope falls within the missing region.
Yes, if its epitope lies within canonical residues 1–96: that region is missing from isoform 2. Confirm the antibody’s mapped epitope and its numbering convention before interpreting absence of a lower band as absence of isoform 2.
Yes, if its epitope lies within canonical residues 1–96: that region is missing from isoform 2. Confirm the antibody’s mapped epitope and its numbering convention before interpreting absence of a lower band as absence of isoform 2.
Which EHHADH lysines carry listed acetylation or succinylation?
PTM · In canonical UniProt numbering, acetylation is listed at 165, 171, 219, 250, 346, 350, 464, 584, 591 and 710; succinylation at 38, 165, 219, 280, 290, 532, 577, 584, 591, 710 and 722. The two modifications are listed as alternate at 165, 219, 584, 591 and 710. These annotations do not establish a visible band shift.
UniProt lists phosphothreonine at canonical residue 548 and phosphoserine at canonical residue 718. Use these coordinates when checking a site-specific antibody; numbering in an antibody datasheet or paper may follow a different convention. Their presence does not establish a detectable mobility change.
UniProt lists phosphothreonine at canonical residue 548 and phosphoserine at canonical residue 718. Use these coordinates when checking a site-specific antibody; numbering in an antibody datasheet or paper may follow a different convention. Their presence does not establish a detectable mobility change.
Does this guide establish induction of EHHADH?
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 EHHADH Western blot?
Transfer · Use a transfer setup suitable for a protein near the canonical predicted mass of 79.5 kDa. Check transfer with a membrane stain and adjust conditions if signal remains in the gel. UniProt features do not specify a membrane or transfer time.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05757-2 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 EHHADH bands be quantified?
Quantitation · Define which band or bands are being measured, since isoform 2 lacks residues 1–96 and may migrate below the canonical protein. Use the same band definition across samples and normalize for loading. A change in signal alone cannot identify an isoform or a listed modification.
Why might EHHADH migrate away from its predicted mass?
Interpretation · The canonical sequence has a predicted mass of 79.5 kDa. Isoform 2 lacks canonical residues 1–96 and is expected to be smaller. Modified residues are also listed, but their presence alone does not establish a visible shift. No observed band position was supplied, so an apparent mass difference cannot be assessed.
Boster reagents
EHHADH 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-EHHADH Antibody
Cat # A05757-2
Real WB data
Anti-EHHADH Antibody (C-term)
Cat # A05757-3
Real WB data
Anti-PBFE EHHADH Antibody
Cat # A05757-1
Three the supplier anti-EHHADH antibodies list WB images. The captions show cell lysates for A05757-2, mouse liver and kidney lysates for A05757-3, and unspecified cells for A05757-1. These examples document tested contexts; they do not establish performance across all listed species.
Which to pick: For mouse liver or kidney, consider A05757-3, whose WB image uses those tissues. For cell lysates, A05757-2 shows Jurkat, Raw264.7, and H9C2 samples at 1:500. A05757-1 lists Human and Rat reactivity, but its WB caption does not identify the cells.
Source: BosterBio EHHADH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q08426.
- Human Protein Atlas. EHHADH tissue expression.
- PMC7672977 — target-verified WB comparison
- PMC8112926 — target-verified WB comparison
- PMC10834178 — target-verified WB comparison