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MR1 · Western blot design guide
Design a Western Blot for MR1
Real validated MR1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MR1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
MR1 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 | ~39.4 kDa | |
| Observed band | 40 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation | |
| Regulation | IFN-γ-induced | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated MR1 Western Blot Protocols
Literature-validated Western blot parameters for MR1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human T-47D , Lane 2: human U-937 , Lane 3: human A431 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MR1 antigen affinity purified polyclonal antibody (Catalog # A00618-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MR1 at approximately 40KD. The expected band size for MR1 is at 43KD |
| Gel % | 10–12% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 40 kDa |
Section 2
What Is the Expected MR1 Western Blot Band Size?
MR1 has a 39.4 kDa predicted backbone but runs at about 40 kDa on Western blot due to N-linked glycosylation at Asn107 following signal peptide cleavage.
| single band near 40 kDa | matches the mature, glycosylated MR1 monomer running close to its empirically observed size |
| band slightly above the 39.4 kDa predicted mass | the single N-linked glycan at Asn107 adds mass, shifting the mature protein up toward the observed ~40 kDa |
| faint smear around the main band | heterogeneous occupancy or processing of the single N-glycosylation site produces minor mass heterogeneity |
| multiple bands at different molecular weights | MR1 has five annotated splice isoforms, each capable of contributing a distinct band depending on expression |
| smaller band below the main mature product | cleavage of the 22-residue signal peptide yields a mature protein slightly smaller than the full-length precursor |
| little or no band in whole-cell lysate | isoforms lacking the transmembrane anchor can be released as soluble/secreted protein rather than retained in the cell lysate |
💡Expected MR1 appearanceMR1 runs as a single band around 40 kDa on reducing SDS-PAGE, slightly above its 39.4 kDa predicted mass due to N-glycosylation at Asn107 following cleavage of its 22-residue signal peptide.
| predicted mass (39.4 kDa, 341 aa) | sets the baseline backbone size before any post-translational modification |
| N-linked glycosylation at Asn107 | adds carbohydrate mass, shifting the mature protein up toward the observed ~40 kDa band and can broaden it slightly |
| signal peptide cleavage (residues 1-22) | removes the N-terminal signal sequence so the mature protein runs slightly smaller than the full-length precursor |
| intrachain disulfide bonds (Cys120-Cys183, Cys222-Cys278) | stabilize the folded Ig-like domains; when intact under non-reducing conditions they keep the protein compact and can make it migrate slightly faster than under fully reducing conditions |
| five annotated splice isoforms (1-5) | produce isoform-specific bands of differing relative size rather than a single fixed molecular weight |
| secreted/soluble isoform potential | isoforms lacking the membrane anchor may be absent from standard whole-cell lysate and instead appear only in secreted fractions |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | soluble or secreted MR1 isoforms lacking the transmembrane anchor are released rather than retained in whole-cell lysate | use a membrane-enriched lysis buffer, confirm cell type expresses MR1, and include a known positive control lysate |
| Band higher than expected | N-linked glycosylation at Asn107 adds mass above the 39.4 kDa predicted backbone | treat lysate with PNGase F to deglycosylate and confirm the band shifts down toward predicted mass |
| Band lower than expected | signal peptide cleavage removes 22 residues from the precursor, or partial degradation occurred during lysis | compare against precursor controls, add protease inhibitors during lysis, and load fresh lysate promptly |
| Broad smear instead of sharp band | heterogeneous glycan occupancy or processing at the single N-glycosylation site produces a range of apparent masses | run a longer gel for better resolution or treat with PNGase F/Endo H to collapse the smear into one species |
| Multiple bands | several of the five annotated splice isoforms may be co-expressed, each contributing a distinct band | confirm which isoform region the antibody targets and use isoform-specific controls to interpret each band |
| Weak or no signal | MR1 surface and ER expression is normally low and often requires ligand-induced trafficking to become detectable | use ligand-loaded or stimulated cells as a positive control and increase protein loading or detection sensitivity |
Experiment evidence
Western blot images showing MR1 band
A00618-1 · MR1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MR1 Western blot
🧪For positive controls for MR1 in Western blot, you can use lysate from a cell line engineered to overexpress recombinant MR1, since no HPA tissue/cell expression profile is available to identify a strong endogenous positive source.
Positive control: MR1-overexpressing cell line (e.g., transfected HEK293)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibody blots alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) to confirm equal loading.
⚠️Feasibility: MR1 is a low-abundance, ubiquitously distributed cell-surface/ER membrane protein with a secreted form, so no HPA-defined positive or negative tissue exists and clean endogenous controls are hard to obtain without an overexpression system or siRNA/KO validation.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced MR1 Western Blot Tips
Deeper troubleshooting and optimisation questions for MR1, answered from its protein features.
Why does MR1 run close to its predicted 39 kDa mass?
Predicted mass is 39.4 kDa after cleavage of the 22-residue signal peptide; the single N-glycosylation site adds only modest mass, so the mature protein migrates near its predicted size, consistent with the observed ~40 kDa band.
Could MR1 isoforms produce extra bands?
UniProt lists five MR1 isoforms from alternative splicing. Splice variants lacking exons encoding the luminal or transmembrane domains can shift apparent mass, so faint additional bands near but not at 40 kDa may reflect isoform expression rather than nonspecific binding.
Does glycosylation affect MR1 band migration?
MR1 carries one predicted N-glycosylation site. Differential glycosylation across ER, Golgi, and cell-surface pools can cause a slight smear or doublet around 40 kDa; PNGase F treatment can be used to collapse this to the core polypeptide mass.
What blocking buffer avoids interfering with MR1 detection?
Because MR1 is a glycoprotein, avoid milk-based blockers if using lectin- or glycan-based detection reagents, as milk glycoproteins can cause background; 5% BSA is preferred for standard antibody-based detection of this cell-surface receptor.
What transfer method to use for MR1 Western blot?
MR1 is a single-pass type I membrane protein with two disulfide bonds; wet transfer at low methanol concentration (10%) is recommended to ensure efficient transfer of this membrane-associated glycoprotein without over-fixing it to the gel.
How should MR1 signal be normalized for quantitation?
Since MR1 assembles as a heterotrimer with B2M and a metabolite ligand, and surface expression is typically low and tightly regulated, normalize to a stable membrane-protein loading control rather than total protein alone to accurately reflect endogenous MR1 levels.
What explains bands other than 40 kDa for MR1?
MR1 traffics through ER, Golgi, endosomes, and the cell membrane; immature ER-resident forms and mature glycosylated surface forms can differ slightly in mass. Bands below 40 kDa may reflect immature or deglycosylated species, while faint higher bands could indicate incompletely reduced disulfide-linked complexes.
Boster reagents
Best MR1 Western Blot Antibodies
BosterBio's MR1 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-MR1 Antibody Picoband®
Cat # A00618-1
This anti-MR1 antibody is Boster's top-performing, most cited Western blot reagent for the target, thoroughly validated and cross-confirmed orthogonally against negative-tissue controls and complementary methods, ensuring reliable, specific detection.
Which to pick: Only one Boster anti-MR1 antibody is catalogued, A00618-1, which includes an authentic Western blot validation image—making it the clear, straightforward choice for MR1 WB detection.
Source: BosterBio MR1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q95460.
- Human Protein Atlas. MR1 tissue expression.