This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ECHS1 / Enoyl-CoA hydratase, mitochondrial · Western blot design guide
Design a Western Blot for ECHS1
Source-linked ECHS1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ECHS1 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
ECHS1 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 | ~31.4 kDa | |
| Observed band | ~29 kDa | |
| Gel | 5–20% (catalog A04508-1) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
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 | 1 isoform(s) |
Section 1
Source-Linked ECHS1 Western Blot Protocol Options
The A04508-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human Hela, human HT1080, human 293T, human MCF-7, human U-87MG, human CACO-2, human PC-3 (catalog A04508-1) |
| Gel % | 5–20% (catalog A04508-1) |
| Load | 30 ug; reducing conditions (catalog A04508-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04508-1) |
| Membrane | nitrocellulose membrane (catalog A04508-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04508-1) |
| Primary antibody | A04508-1 · 0.5 μg/mL (catalog A04508-1) |
| Primary incubation | overnight at 4°C (catalog A04508-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04508-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04508-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04508-1) |
| Detection | ECL (catalog A04508-1) |
Section 2
What Is the Expected ECHS1 Western Blot Band Size?
ECHS1 is predicted at 31.4 kDa and observed at ~29 kDa; the cause of the difference is not established.
| Band at ~29 kDa | Empirical ECHS1 band in reducing lysates. |
| Band near 31.4 kDa | Could correspond to the predicted full-length protein; verify its identity. |
| Doublet near the expected region | Precursor processing or modification is possible, but distinct forms are unproven. |
| Faint or absent band | Mitochondrial ECHS1 may be poorly represented in the sample. |
💡Expected ECHS1 appearanceUniProt predicts 31.4 kDa, while antibody blots show ~29 kDa; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| UniProt predicted mass | Full-length sequence predicts 31.4 kDa; reducing blots show ~29 kDa. |
| Annotated transit peptide | Processing could reduce mature protein size, but its cleavage site and contribution to migration are unspecified. |
| Phosphothreonine at residue 46 | Could affect mobility; no visible shift is established. |
| Alternate acetylation or succinylation at lysine 101 | Could affect mobility; no visible shift is established. |
| Phosphoserine at residue 114 | Could affect mobility; no visible shift is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Insufficient mitochondrial ECHS1 or poor detection. | Check loading, a mitochondrial marker, and a positive lysate. |
| Band higher than expected | A precursor is possible, but the band identity is uncertain. | Compare with the ~29 kDa control band and verify specificity. |
| Band lower than expected | Transit peptide processing is possible, but its size effect is unknown. | Compare with the empirical ~29 kDa band and verify specificity. |
| Multiple bands | Processing or modification is possible; distinct migrating forms are unproven. | Use a positive control and an independent ECHS1 antibody. |
| Weak or no signal | Low ECHS1 recovery or insufficient detection. | Check mitochondrial recovery, loading, and a positive lysate. |
Experiment evidence
Western blot images showing ECHS1 band
A04508-1 · ECHS1
A04508-1 · ECHS1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ECHS1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ECHS1 in Western blot, you can use adipose tissue lysate.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: No Not-detected tissue is listed, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for ECHS1
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Medium | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ECHS1 Western Blot Tips
Deeper troubleshooting and optimisation questions for ECHS1, answered from its protein features.
How should ECHS1 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 an ECHS1 isoform explain a second band?
Isoforms · The supplied record lists one isoform and no alternative sequence. These features do not support assigning a second band to an ECHS1 isoform.
Which ECHS1 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphothreonine at position 46 and phosphoserine at position 114. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence does not establish a visible band shift.
UniProt lists alternate acetylation or succinylation at lysines 101 and 115, acetylation at 118 and 211, and succinylation at 204. These are UniProt sequence coordinates. The alternate annotations do not mean both modifications occupy the same lysine at once, and the features alone do not predict separate visible bands.
No glycosylation sites are supplied for ECHS1. The record therefore provides no basis to assign an unexpected band to glycosylation; band position alone cannot establish its cause.
UniProt lists alternate acetylation or succinylation at lysines 101 and 115, acetylation at 118 and 211, and succinylation at 204. These are UniProt sequence coordinates. The alternate annotations do not mean both modifications occupy the same lysine at once, and the features alone do not predict separate visible bands.
No glycosylation sites are supplied for ECHS1. The record therefore provides no basis to assign an unexpected band to glycosylation; band position alone cannot establish its cause.
Does this guide establish induction of ECHS1?
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 ECHS1?
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 A04508-1 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.
What should be quantified as the ECHS1 signal?
Quantitation · Use a consistently defined band region around the observed approximately 29 kDa signal, and assess any additional bands separately. The predicted mass is 31.4 kDa, while the supplied features do not establish that every band near either mass is ECHS1.
Why might ECHS1 appear near 29 kDa instead of 31.4 kDa?
Interpretation · The supplied apparent band is approximately 29 kDa, versus a predicted mass of 31.4 kDa. ECHS1 is a mitochondrial matrix protein with a transit-peptide keyword, but no cleavage coordinate is supplied. Do not assign the difference to processing or modification from these features alone.
ECHS1 is annotated as a homohexamer, described as a dimer of trimers. Consider sample conditions when assessing a higher band, but do not identify it as an ECHS1 oligomer from position alone. The listed modifications likewise do not establish its identity.
ECHS1 is annotated as a homohexamer, described as a dimer of trimers. Consider sample conditions when assessing a higher band, but do not identify it as an ECHS1 oligomer from position alone. The listed modifications likewise do not establish its identity.
Boster reagents
ECHS1 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-ECHS1 Antibody Picoband®
Cat # A04508-1
A04508-1 is the listed anti-ECHS1 antibody. Its Western blot image shows an approximately 29 kDa band in eight human cell lysates. Human, mouse, and rat reactivity is listed, but the supplied blot shows human samples only.
Which to pick: A04508-1 is the only listed option and has a Western blot image using human HepG2, HeLa, HT1080, 293T, MCF-7, U-87MG, CACO-2, and PC-3 lysates. No mouse or rat blot examples are supplied.
Source: BosterBio ECHS1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P30084.
- Human Protein Atlas. ECHS1 tissue expression.
- PMC10758025 — target-verified WB comparison