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MT-ND5 · Western blot design guide
Design a Western Blot for MT-ND5
Real validated MT-ND5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MT-ND5 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
MT-ND5 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 | ~67 kDa | |
| Observed band | 67 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Membrane protein mobility shift | |
| Regulation | Not found in UniProt | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated MT-ND5 Western Blot Protocols
Literature-validated Western blot parameters for MT-ND5 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human HepG2 , Lane 3: human Raji . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MT-ND5 antigen affinity purified polyclonal antibody (Catalog # A03488-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MT-ND5 at approximately 67 kDa. The expected band size for MT-ND5 is at 67 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 67 kDa |
Section 2
What Is the Expected MT-ND5 Western Blot Band Size?
MT-ND5 has a 67 kDa predicted mass and runs at the same ~67 kDa empirically, with only modest mobility shifts expected from its highly hydrophobic multi-pass membrane topology.
| single sharp band at ~67 kDa | matches the unmodified predicted mass of the MT-ND5 monomer, consistent with the empirically observed band |
| faint or absent band in low-mitochondria fractions | MT-ND5 is confined to the mitochondrial inner membrane, so cytosolic-only preps or samples with few mitochondria give little signal |
| high-molecular-weight smear or material retained near the well | MT-ND5 is a core subunit locked into the 45-subunit Complex I; incomplete solubilization of this membrane-embedded assembly can leave aggregated complex that fails to resolve |
| band migrating slightly off the 67 kDa position despite correct mass | its multi-pass transmembrane topology is highly hydrophobic and can bind SDS anomalously, shifting apparent mobility a bit from the calculated size |
| no additional isoform-sized bands | only one isoform is annotated, so a single species rather than a ladder of bands is expected |
💡Expected MT-ND5 appearanceMT-ND5 is expected as a single band at ~67 kDa in mitochondria-containing lysate, matching both its unmodified predicted mass and the empirically observed 67 kDa band, since it carries no signal peptide, glycosylation, or disulfide partners.
| predicted mass (UniProt) | sets the ~67 kDa reference size for the unmodified monomer, matching the 67 kDa observed band |
| absence of glycosylation sites | band runs as a single clean species without upward smearing from sugar heterogeneity |
| absence of disulfide-linked dimerization | no higher-mass dimer band is expected regardless of reducing or non-reducing conditions |
| multi-pass transmembrane topology | hydrophobic membrane-embedded structure can cause modest anomalous SDS-PAGE mobility even when the mass is correct |
| single annotated isoform | only one band is expected, with no isoform-driven size variants |
| assembly into the 45-subunit Complex I | incomplete denaturation or solubilization of the native complex can leave high-molecular-weight aggregates or smearing near the well |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MT-ND5 sits in the mitochondrial inner membrane and is poorly extracted without adequate detergent lysis, or the sample has low mitochondrial content | use a lysis buffer strong enough to solubilize mitochondrial membranes and confirm the sample is mitochondria-rich |
| Band higher than expected | incomplete denaturation or residual aggregation of this hydrophobic multi-pass membrane protein still associated with other Complex I subunits | avoid over-boiling, ensure sufficient SDS and reducing agent, and optimize denaturation time for membrane proteins |
| Broad smear instead of sharp band | aggregation-prone hydrophobic transmembrane protein that is incompletely denatured before loading | use fresh reducing agent, avoid boiling at high temperature, and load a denaturation protocol suited to membrane proteins |
| Weak or no signal | low mitochondrial content in the sample or protein degradation during preparation | enrich for a mitochondrial fraction, increase total protein loaded, and use freshly prepared lysate |
| Multiple bands | nonspecific antibody binding to other Complex I subunits or degradation products, since only one MT-ND5 isoform exists | validate antibody specificity with a knockdown or knockout control and add protease inhibitors during lysis |
Experiment evidence
Western blot images showing MT-ND5 band
A03488-2 · MT-ND5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MT-ND5 Western blot
🧪For positive controls for MT-ND5 in Western blot, you can use whole-cell lysate from a mitochondria-containing human cell line such as HeLa, since this mitochondrially encoded respiratory chain subunit is ubiquitously expressed in nucleated cells.
Positive control: HeLa cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Alongside MT-ND5, blot for GAPDH and β-actin and include a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) to confirm even loading.
⚠️Feasibility: No HPA expression data are available and, as a ubiquitous mitochondrially encoded OXPHOS subunit, no tissue is expected to be negative, so use mtDNA-depleted (rho0) cells or an siRNA/CRISPR knockdown as the negative control instead.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced MT-ND5 Western Blot Tips
Deeper troubleshooting and optimisation questions for MT-ND5, answered from its protein features.
Why does MT-ND5 run at its predicted 67 kDa mass?
UniProt lists no glycosylation, no modified residues, and no disulfide bonds for MT-ND5, so there is no PTM-driven mass shift and the observed 67 kDa band matches the predicted mass. As a multi-pass inner-membrane protein it can still show minor mobility anomalies if SDS denaturation is incomplete, but a match to predicted mass is the expected default result.
Does MT-ND5 have multiple isoforms on Western blot?
UniProt annotates only one isoform for MT-ND5, consistent with its mitochondrial DNA origin, which is not subject to nuclear alternative splicing. A single clean band at 67 kDa is expected; additional bands should be interpreted as degradation, aggregation, or off-target signal rather than isoform variation.
Is MT-ND5 expression regulated by a specific pathway?
MT-ND5 is a mitochondrial DNA-encoded core subunit of Complex I with no annotated regulatory PTMs, so its abundance largely tracks overall mitochondrial content and Complex I assembly rather than a discrete inducible signal. Normalize against a mitochondrial mass marker or other Complex I subunits when comparing conditions, instead of expecting acute stimulus-driven induction.
How should blocking be optimized for MT-ND5 detection?
As a hydrophobic multi-pass transmembrane protein of the inner mitochondrial membrane, MT-ND5 has no extracellular glycans or disulfides to protect, so standard 5% non-fat milk or BSA blocking is sufficient. Avoid prolonged over-blocking, which can reduce antibody access to membrane-embedded epitopes and weaken signal on an already hydrophobic, membrane-restricted target.
What transfer method to use for MT-ND5 Western blot?
Use wet transfer for this 67 kDa multi-pass membrane protein embedded in the mitochondrial inner membrane; include SDS (0.05-0.1%) in the transfer buffer to help mobilize the hydrophobic protein out of the gel, and extend transfer time given its multiple transmembrane helices, which can slow elution compared to soluble proteins of similar size.
How to normalize MT-ND5 for accurate quantitation?
Because MT-ND5 is a core, mitochondrion-restricted Complex I subunit, normalize to a mitochondrial mass marker (e.g., another OXPHOS subunit or mitochondrial membrane protein) rather than a whole-cell housekeeping gene, since total mitochondrial content varies independently of cytosolic protein loading and can confound comparisons across samples.
What explains unexpected bands on MT-ND5 blots?
MT-ND5 carries disease-associated variants (Leber hereditary optic neuropathy, Leigh syndrome, MELAS) that can alter protein stability or complex assembly, potentially producing faint or shifted bands in patient-derived samples. Its multi-pass transmembrane structure also predisposes it to aggregation if not fully denatured, which can appear as high-molecular-weight smearing above the 67 kDa monomer band.
Boster reagents
Best MT-ND5 Western Blot Antibodies
BosterBio's MT-ND5 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-MT-ND5 Antibody Picoband®
Cat # A03488-2
These recommended anti-MT-ND5 antibodies are Boster's best-performing, most-cited reagents, thoroughly validated by Western blot and orthogonally cross-validated against negative-tissue controls and complementary methods, giving you confidence in specific, reproducible detection of MT-ND5 in your samples.
Which to pick: Only one Boster antibody is catalogued for MT-ND5: A03488-2, backed by an actual Western blot validation image on 5-20% SDS-PAGE; choose it as your default reagent for MT-ND5 detection.
Source: BosterBio MT-ND5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P03915.
- Human Protein Atlas. MT-ND5 tissue expression.