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- Table of Contents
MCAT / Malonyl-CoA-acyl carrier protein transacylase, mitochondrial · Western blot design guide
Design a Western Blot for MCAT
Real validated MCAT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MCAT 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
MCAT 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 | ~43 kDa | |
| Observed band | Approximately 39 kDa | |
| Gel | 10% (catalog A04806-1) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Lymph node (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Observed below predicted | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated MCAT Western Blot Protocols
The A04806-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HepG2, human RT4 (catalog A04806-1) |
| Gel % | 10% (catalog A04806-1) |
| Load | 30 ug; reducing conditions (catalog A04806-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04806-1) |
| Membrane | nitrocellulose membrane (catalog A04806-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04806-1) |
| Primary antibody | A04806-1 · 0.5 μg/mL (catalog A04806-1) |
| Primary incubation | overnight at 4°C (catalog A04806-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04806-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04806-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04806-1) |
| Detection | ECL (catalog A04806-1) |
Section 2
What Is the Expected MCAT Western Blot Band Size?
MCAT is predicted at 43 kDa but observed near 39 kDa; the cause of the difference is not established.
| Band near 39 kDa | Empirical MCAT band in whole-cell lysates; confirm identity with appropriate controls |
| Band near 43 kDa | Near the predicted MCAT mass; identity requires validation |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct migration is unestablished |
| Weak band in whole-cell lysate | Mitochondrial localization may make enrichment useful for detection |
💡Expected MCAT appearanceMCAT has a predicted mass of 43 kDa, while antibody blots show a band near 39 kDa; the difference is unexplained, so confirm band identity with appropriate controls.
| Predicted MCAT mass | 43 kDa is the sequence-based reference; the observed band is near 39 kDa |
| Alternative splicing | Two isoforms are annotated, but their relative masses are unspecified |
| Isoform 1 | Its individual mass and migration relative to isoform 2 are unspecified |
| Isoform 2 | Its individual mass and migration relative to isoform 1 are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial MCAT may be below detection in the lysate | Check loading and antibody performance; try a mitochondrial fraction |
| Band higher than expected | Band identity or migration is uncertain | Compare with the 39 kDa reference and verify specificity by MCAT depletion |
| Band lower than expected | The observed 39 kDa band is below the predicted 43 kDa mass for an unknown reason | Verify specificity by MCAT depletion or immunoprecipitation |
| Multiple bands | The two annotated isoforms are possible contributors, but distinct bands are unproven | Check isoform expression and verify which bands depend on MCAT |
| Weak or no signal | Mitochondrial MCAT may be insufficient in the sample | Check loading and enrich mitochondria |
Experiment evidence
Western blot images showing MCAT band
A04806-1 · MCAT
A04806-1 · MCAT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MCAT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MCAT in Western blot, you can use adipose tissue lysate, which has high HPA expression.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Lymph node (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: MCAT is mitochondrial, and HPA identifies lymph node as a potential negative tissue control.
HPA tissue expression evidence for MCAT
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 → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MCAT Western Blot Tips
Deeper troubleshooting and optimisation questions for MCAT, answered from its protein features.
How should MCAT 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.
How could MCAT isoforms affect the band pattern?
Isoforms · UniProt lists two isoforms. Isoform 2 replaces residues 172–180 and lacks residues 181–390 of the 390-residue canonical sequence. It could therefore produce a smaller band, but the features do not establish its apparent mass or whether it is present in your sample.
Residues 1–171 are shared in the supplied sequences. An antibody against that region could recognize both isoforms. Check the antibody’s actual epitope: residues 172–180 differ in isoform 2, and canonical residues 181–390 are absent from it.
Define whether the measurement targets canonical MCAT or both isoforms. A reagent against canonical residues 181–390 would miss isoform 2; a shared-region reagent could detect both. If bands resolve separately, report which band was measured rather than treating their signals as one species.
Residues 1–171 are shared in the supplied sequences. An antibody against that region could recognize both isoforms. Check the antibody’s actual epitope: residues 172–180 differ in isoform 2, and canonical residues 181–390 are absent from it.
Define whether the measurement targets canonical MCAT or both isoforms. A reagent against canonical residues 181–390 would miss isoform 2; a shared-region reagent could detect both. If bands resolve separately, report which band was measured rather than treating their signals as one species.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of MCAT?
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 MCAT?
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 A04806-1 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.
How should MCAT be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might MCAT run near 39 kDa instead of its predicted 43 kDa?
Interpretation · Approximately 39 kDa is the observed band, while 43 kDa is the predicted mass. MCAT is mitochondrial and has a transit-peptide keyword, but the supplied features give no cleavage site or mature mass. These features do not establish the cause of the difference; confirm the band’s identity before assigning it to processing.
The supplied modification is N6-succinyllysine at UniProt position 314. That position is absent from isoform 2. Its presence in the feature list does not show that a modified species produces a visible shift or explain the approximately 39-kDa band. Keep UniProt numbering explicit when comparing antibody or paper annotations.
Isoform 2 is a candidate because it lacks canonical residues 181–390. Band size alone cannot identify it. Compare detection with antibodies against shared residues 1–171 and canonical residues 181–390, while keeping the antibody epitopes and the observed approximately 39-kDa band in view.
The supplied modification is N6-succinyllysine at UniProt position 314. That position is absent from isoform 2. Its presence in the feature list does not show that a modified species produces a visible shift or explain the approximately 39-kDa band. Keep UniProt numbering explicit when comparing antibody or paper annotations.
Isoform 2 is a candidate because it lacks canonical residues 181–390. Band size alone cannot identify it. Compare detection with antibodies against shared residues 1–171 and canonical residues 181–390, while keeping the antibody epitopes and the observed approximately 39-kDa band in view.
Boster reagents
MCAT 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-MCAT Antibody Picoband®
Cat # A04806-1
The catalog reports one anti-MCAT antibody, A04806-1. Its Western blot image shows a band near 39 kDa in four human whole-cell lysates; the expected size is 43 kDa. The supplied image does not demonstrate mouse or rat Western blot performance.
Which to pick: A04806-1 is the only listed option. It lists human, mouse, and rat reactivity, with a Western blot image from human HeLa, HepG2, RT4, and 293T lysates.
Source: BosterBio MCAT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8IVS2.
- Human Protein Atlas. MCAT tissue expression.
- PMC5650417 — target-verified WB comparison
- PMC5028732 — target-verified WB comparison