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- Table of Contents
ME3 / NADP-dependent malic enzyme, mitochondrial · Western blot design guide
Design a Western Blot for ME3
Real validated ME3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ME3 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
ME3 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 | ~67.1 kDa | |
| Observed band | ~67 kDa | |
| Gel | 5–20% (catalog A05164-2) | |
| Positive control | Caudate (IHC candidate; verify WB) +4 more | |
| Negative control | Bone marrow (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated ME3 Western Blot Protocols
The A05164-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | monkey COS7, human HEL, rat kidney (catalog A05164-2) |
| Gel % | 5–20% (catalog A05164-2) |
| Load | 30 ug; reducing conditions (catalog A05164-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05164-2) |
| Membrane | nitrocellulose membrane (catalog A05164-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05164-2) |
| Primary antibody | A05164-2 · 0.25 μg/mL (catalog A05164-2) |
| Primary incubation | overnight at 4°C (catalog A05164-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05164-2) |
| Secondary incubation | 1.5 hour at RT (catalog A05164-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05164-2) |
| Detection | ECL (catalog A05164-2) |
Section 2
What Is the Expected ME3 Western Blot Band Size?
ME3 is predicted at 67.1 kDa and observed at ~67 kDa; the cause of their small difference is not established.
| Band at ~67 kDa | Matches the empirical ME3 band. |
| Single band near 67.1 kDa | Consistent with the UniProt predicted mass. |
| Additional band at a different mass | Could reflect isoform 1 or 2; their migration is not established. |
| Little or no band in a cytosolic fraction | ME3 is located in the mitochondrial matrix. |
💡Expected ME3 appearanceME3 is predicted at 67.1 kDa and observed at ~67 kDa in reducing whole-cell blots; confirm band identity with an orthogonal antibody or ME3 depletion.
| UniProt predicted mass | Places the reference band near 67.1 kDa. |
| 604-amino-acid sequence | Underlies the reported whole-protein mass estimate. |
| Isoform 1 | May differ in size from isoform 2; its individual mass is not supplied. |
| Isoform 2 | May differ in size from isoform 1; its individual mass is not supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | The sample may contain too little mitochondrial ME3. | Check mitochondrial content and loading; compare with a mitochondrial-enriched fraction. |
| Band higher than expected | Its identity or relationship to the listed isoforms is unconfirmed. | Compare with the ~67 kDa band and test an orthogonal antibody or ME3 depletion. |
| Band lower than expected | Its identity or relationship to the listed isoforms is unconfirmed. | Check sample integrity and confirm the band by ME3 depletion. |
| Multiple bands | Isoforms 1 and 2 are listed, but distinct migration is unproven. | Confirm each band with isoform-aware controls or ME3 depletion. |
| Weak or no signal | Low ME3 abundance in the sampled material may limit detection. | Check loading and compare with a mitochondrial-enriched fraction. |
Experiment evidence
Western blot images showing ME3 band
A05164-2 · ME3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ME3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ME3 in Western blot, you can use caudate tissue, which HPA reports as highly positive.
Positive control: Caudate (IHC candidate; verify WB)
Negative control: Bone marrow (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: ME3 is in the mitochondrial matrix, so use lysate that retains mitochondrial proteins.
HPA tissue expression evidence for ME3
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ME3 Western Blot Tips
Deeper troubleshooting and optimisation questions for ME3, answered from its protein features.
How should ME3 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 ME3 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residues 309–342 are replaced and residues 343–604 are missing, so a shorter band is plausible. The supplied features do not establish its apparent mass or whether it is detected in your sample.
Check the antibody epitope against both sequences. An epitope within canonical residues 343–604 is absent from isoform 2; residues 309–342 are replaced in isoform 2. An antibody targeting either region may detect the isoforms differently.
Check the antibody epitope against both sequences. An epitope within canonical residues 343–604 is absent from isoform 2; residues 309–342 are replaced in isoform 2. An antibody targeting either region may detect the isoforms differently.
What does ME3 phosphorylation imply for band interpretation?
PTM · UniProt annotates phosphoserine at position 371 in the canonical sequence. That region is missing from isoform 2. A phosphorylation dependent band shift is possible, but the site annotation alone does not demonstrate a visible shift.
Does this guide establish induction of ME3?
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 ME3?
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 A05164-2 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 can ME3 bands be quantified across samples?
Quantitation · Quantify the approximately 67 kDa band consistently across samples. If additional bands appear, assess them separately until their identities are established; isoform 2 has a substantially different sequence, and phosphorylation at canonical Ser371 does not by itself establish a distinct band.
Does the observed ME3 band match its predicted mass?
Interpretation · The observed band at approximately 67 kDa agrees with the 67.1 kDa predicted mass. Apparent migration can vary, so this agreement alone does not establish band identity.
ME3 is listed in the mitochondrial matrix and carries a transit peptide keyword, but no cleavage position or processed mass is supplied. Do not assign a lower band to mitochondrial processing from these features alone.
Compare bands with the approximately 67 kDa observed band and the annotated isoform changes. A lower band could be consistent with isoform 2, but size alone cannot identify it. Consider whether the antibody epitope is retained in that isoform.
ME3 is listed in the mitochondrial matrix and carries a transit peptide keyword, but no cleavage position or processed mass is supplied. Do not assign a lower band to mitochondrial processing from these features alone.
Compare bands with the approximately 67 kDa observed band and the annotated isoform changes. A lower band could be consistent with isoform 2, but size alone cannot identify it. Consider whether the antibody epitope is retained in that isoform.
Boster reagents
ME3 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-ME3 Antibody Picoband®
Cat # A05164-2
The listed anti-ME3 antibody, A05164-2, has a Western blot image showing an approximately 67 kDa band in monkey COS7 cells, human HEL cells, and rat and mouse tissues. No publication or independent validation evidence is supplied.
Which to pick: A05164-2 is the only listed option. It reports human, monkey, mouse, and rat reactivity; its WB image shows specific cell and tissue lysates from each. Compare your sample and conditions with those shown when deciding whether it fits your experiment.
Source: BosterBio ME3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16798.
- Human Protein Atlas. ME3 tissue expression.
- PMC13551346 — target-verified WB comparison
- PMC11364750 — target-verified WB comparison