This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
MUC5B / Mucin-5B · Western blot design guide
Design a Western Blot for MUC5B
Real validated MUC5B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MUC5B 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
MUC5B 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 | ~596.3 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Positive control | Appendix (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 | Glycosylated + Cleaved | |
| Caveat | Disulfide-linked multimers | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated MUC5B Western Blot Protocols
The M00719 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Hela cell lysate (catalog M00719) |
| Gel % | 4–12% gradient (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M00719; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| 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 MUC5B Western Blot Band Size?
MUC5B has a predicted full-length mass of 596.3 kDa; glycosylation, signal-peptide cleavage, and disulfide-linked multimers may affect migration, but no observed band is supplied.
| Band near 596.3 kDa | compatible with the predicted full-length polypeptide, pending band-identity controls |
| Band above 596.3 kDa or diffuse high-mass signal | compatible with N-linked glycosylation, though its visible effect is unestablished |
| Very high-mass band under non-reducing conditions | compatible with disulfide-linked MUC5B multimers |
| Band slightly below the full-length precursor | compatible with signal-peptide cleavage |
| Little or no band in whole-cell lysate | compatible with secretion of MUC5B |
💡Expected MUC5B appearanceThe predicted full-length polypeptide is 596.3 kDa; no empirical band size is supplied, and glycosylation, signal-peptide cleavage, and disulfide-linked assembly may affect migration, so confirm band identity with controls.
| Predicted full-length mass | sets a 596.3 kDa polypeptide reference, not a validated native band |
| N-linked glycosylation at Asn145 and other annotated sites | may increase apparent size or broaden migration; the extent is unknown |
| Disulfide-linked homomultimer formation | may produce bands above the monomer under non-reducing or incompletely reducing conditions |
| Signal-peptide cleavage at residues 1–25 | makes the mature chain slightly smaller than the full-length precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MUC5B is secreted | examine conditioned medium and include a positive control |
| Band higher than expected | N-linked glycosylation or disulfide-linked multimerization may contribute | compare reducing conditions and assess glycosylation with appropriate controls |
| Band lower than expected | signal-peptide cleavage may slightly reduce chain size | confirm identity with an independent antibody or target depletion |
| Broad smear instead of sharp band | variation across annotated N-linked glycosylation sites may affect migration | compare with a controlled deglycosylation sample and verify specificity |
| Multiple bands | disulfide-linked multimers and glycosylated chains may migrate differently | compare reducing and non-reducing samples and confirm target identity |
| Weak or no signal | secretion may leave little MUC5B in the sampled lysate | test conditioned medium alongside a positive control |
Experiment evidence
Western blot images showing MUC5B band
M00719 · MUC5B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MUC5B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MUC5B in Western blot, you can use appendix tissue.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Because MUC5B is secreted, whole-tissue lysate may have limited signal; consider a secreted fraction.
HPA tissue expression evidence for MUC5B
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 |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MUC5B Western Blot Tips
Deeper troubleshooting and optimisation questions for MUC5B, answered from its protein features.
How should MUC5B 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 different MUC5B isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign multiple bands to annotated isoforms on this evidence alone.
Which annotated modifications matter when interpreting MUC5B bands?
PTM · The record lists 38 glycosylation sites, including N-linked Asn145 and C-linked Trp1340, using UniProt sequence coordinates. Check the numbering convention before comparing these positions with antibody or paper coordinates. The annotations do not establish which modifications occur in a particular sample or whether they produce a visible shift.
Does this guide establish induction of MUC5B?
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.
What transfer method to use for MUC5B Western blot?
Transfer · Plan for a protein with a predicted mass of 596.3 kDa and disulfide-linked higher-order assemblies. Optimize gel and transfer conditions for high-mass material, then check the gel and membrane for retained protein. The supplied features do not establish one transfer method as best.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00719 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 MUC5B 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.
Should MUC5B migrate at its predicted 596.3 kDa?
Interpretation · Use 596.3 kDa as the predicted mass of the 5,762-residue sequence, not an expected band position. MUC5B has annotated glycosylation and forms disulfide-linked multimers, but these features alone cannot predict its apparent mass or establish a band shift.
MUC5B is annotated as secreted. For a secretion comparison, measure the relevant extracellular fraction and keep collection and loading conditions consistent across samples. A cell lysate alone may not represent the secreted pool.
MUC5B is annotated as a disulfide-linked homomultimer with 17 disulfide bonds. Compare matched reducing and nonreducing samples when assessing high-mass signal. Band position alone cannot identify a multimer or another MUC5B species.
The supplied sequence has a signal peptide at residues 1–25. When selecting an antibody, check whether its epitope lies within that interval; a signal-peptide epitope may not report the secreted protein. Do not infer a measurable band shift from this annotation alone.
MUC5B is annotated as secreted. For a secretion comparison, measure the relevant extracellular fraction and keep collection and loading conditions consistent across samples. A cell lysate alone may not represent the secreted pool.
MUC5B is annotated as a disulfide-linked homomultimer with 17 disulfide bonds. Compare matched reducing and nonreducing samples when assessing high-mass signal. Band position alone cannot identify a multimer or another MUC5B species.
The supplied sequence has a signal peptide at residues 1–25. When selecting an antibody, check whether its epitope lies within that interval; a signal-peptide epitope may not report the secreted protein. Do not infer a measurable band shift from this annotation alone.
Boster reagents
MUC5B 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-MUC5B Monoclonal Antibody
Cat # M00719
The catalog reports one anti-MUC5B antibody, M00719, with reported human reactivity. Its Western blot image shows MUC5B expression in HeLa cell lysate; the supplied evidence describes this sample context only.
Which to pick: M00719 is the only listed option. It has a Western blot image from HeLa cell lysate and reported human reactivity; assess suitability for other samples separately.
Source: BosterBio MUC5B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9HC84.
- Human Protein Atlas. MUC5B tissue expression.
- PMC10756985 — target-verified WB comparison
- PMC5624091 — target-verified WB comparison
- PMC4047408 — target-verified WB comparison