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- Table of Contents
MANF / Mesencephalic astrocyte-derived neurotrophic factor · Western blot design guide
Design a Western Blot for MANF
Source-linked MANF Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MANF 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
MANF 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 | ~20.7 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Bone marrow (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Cleaved | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked MANF Western Blot Protocol Options
The A03825 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | 293 cells (catalog A03825) |
| 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 | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A03825; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | 1:20000 (catalog A03825) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected MANF Western Blot Band Size?
MANF has a predicted 20.7 kDa precursor; signal-peptide cleavage may yield a smaller form, but its migration has not been demonstrated here.
| Band near 20.7 kDa | consistent with the predicted precursor size; identity needs confirmation |
| Band below 20.7 kDa | could reflect cleavage of the 1–24 signal peptide |
| Precursor and smaller band | could reflect uncleaved and signal-cleaved MANF |
| Little or no band in whole-cell lysate | consistent with secretion, though MANF is also found in the ER lumen |
💡Expected MANF appearanceUniProt predicts a 20.7 kDa MANF precursor; signal-peptide cleavage could yield a smaller mature band, but no empirical band size is supplied, so confirm identity with appropriate controls.
| Predicted precursor mass | sets a 20.7 kDa reference for the full-length protein |
| Signal peptide at residues 1–24 | cleavage can lower the mature protein's mass |
| Uncleaved signal peptide | retention would leave the precursor larger than the cleaved form |
| Precursor and mature forms | coexistence could produce bands at different positions; their migration is unverified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MANF can be secreted | check conditioned medium alongside lysate |
| Band higher than expected | an uncleaved precursor may run above the mature form | compare lysate and conditioned medium and confirm band identity |
| Band lower than expected | the signal peptide may have been cleaved | compare with a full-length MANF control |
| Multiple bands | precursor and mature MANF may coexist | compare fractions and verify which bands are MANF |
| Weak or no signal | secretion may reduce MANF in the sampled lysate | check conditioned medium and include a positive control |
Experiment evidence
Western blot images showing MANF band
A03825 · MANF
A03825 · MANF
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MANF Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MANF in Western blot, you can use bone marrow lysate, which has high HPA expression.
Positive control: Bone marrow (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: MANF is secreted and retained in the ER, so consider testing conditioned medium alongside lysate.
HPA tissue expression evidence for MANF
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular 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 → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MANF Western Blot Tips
Deeper troubleshooting and optimisation questions for MANF, answered from its protein features.
How should MANF 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 MANF isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. These features provide no basis for assigning multiple bands to MANF isoforms.
How should MANF phosphorylation be assessed?
PTM · The listed modification is phosphotyrosine at UniProt residue 76. If the 1–24 signal peptide is removed, that residue corresponds to position 52 in the processed sequence. State which numbering convention an antibody uses. This site alone does not establish a visible mobility shift.
Does this guide establish induction of MANF?
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 MANF?
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 A03825 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 MANF be quantified across samples?
Quantitation · MANF is listed in the ER lumen and as secreted. Compare like sample fractions across conditions, and report whether measurements come from cell material or extracellular medium. The listed features do not establish how much MANF appears in either fraction.
Why might MANF migrate differently from its predicted 20.7 kDa?
Interpretation · The 20.7 kDa prediction includes the 182-residue precursor. MANF has a signal peptide at residues 1–24, so processing could lower the mass of the detected protein. No observed band size is supplied; the feature alone cannot establish the apparent mass or explain a specific difference.
Check whether the antibody recognizes a sequence retained after removal of the 1–24 signal peptide. MANF is listed in the ER lumen and as secreted, so the sample type may affect which form is detected.
MANF has four listed disulfide bonds. Compare reducing and nonreducing sample preparation when investigating differing band patterns, and keep preparation conditions consistent across samples. The bond count alone does not predict a particular band position.
Consider signal-peptide processing, the listed phosphotyrosine at UniProt residue 76, and the four disulfide bonds alongside sample preparation. The record lists one isoform. Without an observed band size or band-identification evidence, these features cannot assign an unexpected band to a particular MANF form.
Check whether the antibody recognizes a sequence retained after removal of the 1–24 signal peptide. MANF is listed in the ER lumen and as secreted, so the sample type may affect which form is detected.
MANF has four listed disulfide bonds. Compare reducing and nonreducing sample preparation when investigating differing band patterns, and keep preparation conditions consistent across samples. The bond count alone does not predict a particular band position.
Consider signal-peptide processing, the listed phosphotyrosine at UniProt residue 76, and the four disulfide bonds alongside sample preparation. The record lists one isoform. Without an observed band size or band-identification evidence, these features cannot assign an unexpected band to a particular MANF form.
Boster reagents
MANF 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-ARP Antibody
Cat # A03825
The catalog reports A03825 (Anti-ARP Antibody) for MANF, with reported human, mouse, and rat reactivity. Its WB image shows analysis of 293 cells with secondary antibody diluted 1:20000; that example alone does not establish WB performance across all listed species.
Which to pick: A03825 is the only listed option and has a WB image from 293 cells. Check that its reported reactivity covers your sample species; the supplied evidence does not show WB results for other specimens.
Source: BosterBio MANF gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P55145.
- Human Protein Atlas. MANF tissue expression.
- PMC5832864 — target-verified WB comparison
- PMC7415242 — target-verified WB comparison
- PMC10719710 — target-verified WB comparison