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- Table of Contents
MLN / Promotilin · Western blot design guide
Design a Western Blot for MLN
Real validated MLN Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MLN 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
MLN 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 | ~12.9 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Duodenum (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 | Cleaved | |
| Caveat | Processing-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated MLN Western Blot Protocols
The A01292 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A01292) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A01292) |
| Primary antibody | A01292 · 1:1000 (catalog A01292) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A01292) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A01292) |
Section 2
What Is the Expected MLN Western Blot Band Size?
MLN has a predicted full-length mass of 12.9 kDa; signal-peptide cleavage and isoforms may affect migration, but no band position is empirically established.
| Band near 12.9 kDa | consistent with the predicted full-length precursor; identity requires confirmation |
| Band below 12.9 kDa | could reflect removal of the signal peptide at residues 1–25 |
| Little or no band in cell lysate | MLN is secreted and may be found in the medium |
| Multiple bands | could reflect isoforms 1, 2, and 3, whose migration is unknown |
💡Expected MLN appearanceThe predicted full-length mass is 12.9 kDa; signal-peptide removal may yield a smaller band, but no empirical band size is supplied, so confirm band identity with appropriate controls.
| Predicted full-length mass | 12.9 kDa is the sequence-based reference, not a measured band position |
| Signal peptide at residues 1–25 | its removal can make the mature protein smaller than the precursor |
| Isoform 1 | its apparent size relative to the other isoforms is unknown |
| Isoforms 2 and 3 | their apparent sizes relative to isoform 1 and each other are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MLN is secreted | test conditioned medium alongside the lysate |
| Band lower than expected | signal-peptide removal may reduce size | compare precursor and secreted samples and confirm band identity |
| Multiple bands | alternative splicing produces isoforms 1, 2, and 3, though distinct bands are unproven | check antibody specificity and compare samples with different MLN expression |
| Weak or no signal | secretion may leave little MLN in the sampled lysate | test conditioned medium and include a positive control |
| Fragments below expected size | cleavage at paired basic residues may generate smaller products | confirm the fragments with an independent antibody or MLN depletion control |
Experiment evidence
Western blot images showing MLN band
A01292 · MLN
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MLN Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MLN in Western blot, you can use duodenum tissue, where HPA reports high expression.
Positive control: Duodenum (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Because MLN is secreted, whole-cell lysates may have weak signal; conditioned medium may be more suitable.
HPA tissue expression evidence for MLN
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 |
|---|---|---|---|---|
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | endocrine cells | High | 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 | endocrine 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 MLN Western Blot Tips
Deeper troubleshooting and optimisation questions for MLN, answered from its protein features.
How should MLN 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.
Which MLN isoforms could affect band interpretation?
Isoforms · UniProt lists three isoforms. In isoform 2, residues 106–113 change from SEMLPQHA to T; in isoform 3, residues 114–115 change from AK to K. These are UniProt precursor coordinates. The sequence differences may affect antibody recognition or migration, but the features do not establish whether the isoforms resolve as separate bands.
An antibody targeting the common sequence is better suited to comparing total MLN across isoforms. An epitope spanning UniProt residues 106–113 or 114–115 may recognize the listed isoforms differently; verify the antibody’s target sequence before interpreting band intensity.
An antibody targeting the common sequence is better suited to comparing total MLN across isoforms. An epitope spanning UniProt residues 106–113 or 114–115 may recognize the listed isoforms differently; verify the antibody’s target sequence before interpreting band intensity.
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 MLN?
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 MLN?
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 A01292 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 MLN bands be quantified?
Quantitation · Define which MLN form the antibody measures and quantify that form consistently across samples. Because UniProt lists secretion, a signal peptide, processing, and three isoforms, combining distinct bands or comparing different sample fractions could change what the measurement represents.
Why might MLN migrate differently from its predicted 12.9 kDa mass?
Interpretation · The 12.9 kDa prediction is for the 115-residue precursor. UniProt lists a signal peptide at residues 1–25 and identifies MLN as a secreted hormone with cleavage on a pair of basic residues. Processing could produce forms unlike the full precursor, but these features alone do not establish a visible shift or explain any particular band.
Check whether the antibody recognizes the signal peptide at residues 1–25 or a region expected to remain after processing. UniProt also flags cleavage on a pair of basic residues but supplies no cleavage position here, so do not assume which additional residues a processed product retains.
UniProt identifies MLN as secreted. Consider the extracellular or conditioned-medium fraction when selecting a sample, and specify which fraction was tested when interpreting a weak or absent signal.
Compare each band with the 12.9 kDa precursor prediction, the signal peptide at residues 1–25, and the listed isoform sequence changes. UniProt also flags cleavage on a pair of basic residues. These features suggest possibilities to check, but none identifies an unexpected band by itself; apparent band size was not supplied.
Check whether the antibody recognizes the signal peptide at residues 1–25 or a region expected to remain after processing. UniProt also flags cleavage on a pair of basic residues but supplies no cleavage position here, so do not assume which additional residues a processed product retains.
UniProt identifies MLN as secreted. Consider the extracellular or conditioned-medium fraction when selecting a sample, and specify which fraction was tested when interpreting a weak or absent signal.
Compare each band with the 12.9 kDa precursor prediction, the signal peptide at residues 1–25, and the listed isoform sequence changes. UniProt also flags cleavage on a pair of basic residues. These features suggest possibilities to check, but none identifies an unexpected band by itself; apparent band size was not supplied.
Boster reagents
MLN 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-Promotilin MLN Antibody
Cat # A01292
The catalog reports A01292, an anti-MLN antibody with stated human and mouse reactivity. Its Western blot image uses extracts from various cell lines at a 1:1000 primary dilution and 25 µg per lane; the caption does not identify the cell lines.
Which to pick: A01292 is the only listed option and has a Western blot image. Check its stated human or mouse reactivity against your sample; the image caption does not identify which cell lines were tested.
Source: BosterBio MLN gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P12872.
- Human Protein Atlas. MLN tissue expression.
- PMC8051712 — target-verified WB comparison