This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
MEST / Mesoderm-specific transcript homolog protein · Western blot design guide
Design a Western Blot for MEST
Real validated MEST Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MEST 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
MEST 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 | ~38.8 kDa | |
| Observed band | ~43 kDa | |
| Gel | 5–20% (catalog A07241-1) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated MEST Western Blot Protocols
The A07241-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Caco-2, human MCF-7, human Hela (catalog A07241-1) |
| Gel % | 5–20% (catalog A07241-1) |
| Load | 30 ug; reducing conditions (catalog A07241-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A07241-1) |
| Membrane | nitrocellulose membrane (catalog A07241-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A07241-1) |
| Primary antibody | A07241-1 · 0.5 μg/mL (catalog A07241-1) |
| Primary incubation | overnight at 4°C (catalog A07241-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A07241-1) |
| Secondary incubation | 1.5 hour at RT (catalog A07241-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A07241-1) |
| Detection | ECL (catalog A07241-1) |
Section 2
What Is the Expected MEST Western Blot Band Size?
MEST is predicted at 38.8 kDa and observed near 43 kDa; the cause of the difference is not established.
| Band near 43 kDa | Empirical MEST band reported in whole-cell lysates; confirm identity with antibody controls |
| Band near 38.8 kDa | Migration near the predicted MEST mass |
| Band above 38.8 kDa | Asn163 N-linked glycosylation could affect migration, but its contribution is unestablished |
| Multiple bands | Isoforms 1, 2 and 3 could contribute; distinct migration is unestablished |
| Weak or absent band in whole-cell lysate | ER membrane localization may limit recovery during extraction |
💡Expected MEST appearanceMEST has a predicted mass of 38.8 kDa and an empirical band near 43 kDa; the cause of the difference is unestablished, so confirm band identity with antibody controls.
| Predicted MEST mass | 38.8 kDa provides the sequence-based reference; the reported empirical band is near 43 kDa |
| N-linked glycosylation at Asn163 | Could alter apparent mass; its contribution to the 43 kDa band is unestablished |
| Splice isoform 1 | May migrate differently from other isoforms; its individual mass is unknown |
| Splice isoform 2 | May migrate differently from other isoforms; its individual mass is unknown |
| Splice isoform 3 | May migrate differently from other isoforms; its individual mass is unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ER membrane protein may be poorly recovered | Check membrane extraction and include a suitable positive control |
| Band higher than expected | Asn163 N-linked glycosylation could affect migration; the cause of the reported 43 kDa band is unestablished | Compare with a deglycosylated sample and confirm antibody specificity |
| Band lower than expected | An alternative splice isoform is possible, but its mass is unknown | Check isoform expression and verify band identity |
| Multiple bands | MEST has isoforms 1, 2 and 3, though distinct bands are unestablished | Compare isoform controls and check antibody specificity |
| Weak or no signal | Recovery of ER membrane-associated MEST may be low | Optimize membrane solubilization and check a positive control |
Experiment evidence
Western blot images showing MEST band
A07241-1 · MEST
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MEST Western blot
🧪For positive controls for MEST in Western blot, you can use a sample with independently verified MEST expression; no HPA positive tissue or cell was supplied.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: MEST is an ER multipass membrane protein, and the supplied HPA data cannot establish tissue controls.
HPA tissue expression evidence for MEST
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced MEST Western Blot Tips
Deeper troubleshooting and optimisation questions for MEST, answered from its protein features.
How should MEST 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 MEST isoforms could affect band interpretation?
Isoforms · UniProt lists isoforms 1, 2, and 3. Relative to the canonical sequence, isoforms 2 and 3 lack residues 1–9; isoform 3 also lacks residues 218–251. These sequence differences may affect migration or antibody recognition, but they do not establish which isoform produced a band.
How should MEST glycosylation be assessed?
PTM · UniProt lists one N-linked glycosylation site at Asn163 in canonical-sequence numbering. Compare matched untreated and deglycosylated samples when testing whether glycosylation affects migration. A changed band would support that interpretation; the listed site alone does not prove a visible shift.
Does this guide establish induction of MEST?
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 MEST Western blot?
Transfer · MEST is a multi-pass endoplasmic reticulum membrane protein with a predicted mass of 38.8 kDa and an observed band near 43 kDa. Choose transfer conditions that retain proteins in this size range and check transfer with a total-protein stain. The supplied features do not specify one optimal transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A07241-1 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 MEST bands be quantified?
Quantitation · Quantify the same defined band across samples and normalize to total protein. Record whether the band is near the observed 43 kDa position. Because UniProt lists three isoforms and an N-linked glycosylation site, avoid combining distinct bands without establishing what each represents.
Why might MEST appear near 43 kDa instead of 38.8 kDa?
Interpretation · The observed band is about 43 kDa, while the predicted mass is 38.8 kDa. UniProt lists N-linked glycosylation at Asn163 and three isoforms, but these features alone do not establish the cause of the difference. Confirm band identity before assigning the shift to glycosylation.
Consider the listed isoforms and glycosylation at canonical Asn163 when investigating additional bands. Isoform 3 lacks residues 1–9 and 218–251; isoform 2 lacks residues 1–9. Neither feature alone identifies an unexpected band, so verify its identity before assigning it to an isoform or glycosylation state.
Consider the listed isoforms and glycosylation at canonical Asn163 when investigating additional bands. Isoform 3 lacks residues 1–9 and 218–251; isoform 2 lacks residues 1–9. Neither feature alone identifies an unexpected band, so verify its identity before assigning it to an isoform or glycosylation state.
Boster reagents
MEST 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-MEST Antibody Picoband®
Cat # A07241-1
The catalog reports one anti-MEST antibody, A07241-1, with stated human, mouse, and rat reactivity. Its Western blot image shows a band near 43 kDa, versus an expected 40 kDa, in the specified cell and testis lysates. No independent specificity validation is supplied.
Which to pick: A07241-1 is the only listed option. Its WB image includes human Caco-2, MCF-7, HeLa, and RT4 cell lysates plus rat and mouse testis lysates, tested at 30 µg per lane under reducing conditions with 0.5 µg/mL primary antibody.
Source: BosterBio MEST gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q5EB52.
- Human Protein Atlas. MEST tissue expression.
- PMC8798353 — target-verified WB comparison
- PMC5788725 — target-verified WB comparison
- PMC6382817 — target-verified WB comparison