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PRLR · Western blot design guide
Design a Western Blot for PRLR
Real validated PRLR Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PRLR WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
PRLR 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 | ~69.5 kDa | |
| Observed band | 65-95 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 | N-glycosylation increases mass | |
| Regulation | Estrogen up | |
| Isoform | 9 isoform(s) |
Section 1
Real Curated PRLR Western Blot Protocols
Literature-validated Western blot parameters for PRLR — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human MCF-7 . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-PRLR antigen affinity purified polyclonal antibody (Catalog # PB9782) 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 1:5000 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 PRLR at approximately 65, 95 kDa. The expected band size for PRLR is at 70 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| 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, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 65, 95 kDa |
Section 2
What Is the Expected PRLR Western Blot Band Size?
PRLR has a 69.5 kDa predicted backbone but runs at 65-95 kDa on Western blot due to heavy N-glycosylation at three extracellular sites and splice-isoform diversity.
| diffuse band spanning roughly 65-95 kDa | mature PRLR monomer carrying variable N-linked glycan load on top of the 69.5 kDa unmodified backbone |
| broad or smeared band rather than one sharp line | heterogeneous glycan occupancy/processing across the three extracellular N-glycosylation sites (Asn59, Asn104, Asn233) |
| fainter band near 69-70 kDa alongside the main band | a less-glycosylated or precursor-like PRLR species closer to the predicted unmodified mass |
| multiple distinct bands at different molecular weights | reflects the 9 annotated PRLR splice isoforms, which include both membrane-bound and soluble/secreted forms |
| no doubled band at roughly twice the monomer size under reducing conditions | PRLR homodimerizes non-covalently upon prolactin binding; its annotated disulfides are intrachain, not inter-chain, so no covalent dimer survives reducing SDS-PAGE |
💡Expected PRLR appearancePRLR characteristically appears as a broad, heterogeneously glycosylated band at 65-95 kDa on reducing SDS-PAGE, well above the 69.5 kDa unmodified backbone predicted from its 622-residue sequence.
| Predicted mass from UniProt | the 622-aa PRLR backbone predicts a 69.5 kDa protein before any post-translational modification |
| N-glycosylation at Asn59, Asn104, Asn233 | adds variable glycan mass that shifts and broadens the apparent band up to the observed 65-95 kDa range |
| Signal peptide cleavage (residues 1-24) | removal of the signal peptide slightly reduces the mature membrane-bound protein's mass relative to the unprocessed precursor |
| Alternative splicing (9 named isoforms) | generates protein species of differing length, including membrane-bound and soluble/secreted forms, seen as additional bands |
| Non-covalent, hormone-induced homodimerization | because the annotated disulfides are intrachain rather than inter-chain, the ligand-induced dimer dissociates under reducing SDS-PAGE, so no covalent dimer band is expected |
| Intrachain disulfide bonds (Cys36-Cys46, Cys75-Cys86) | stabilize extracellular domain folding; incomplete reduction can subtly alter migration without producing a dimer-sized band |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | extensive N-glycosylation at the three extracellular asparagines raises the apparent mass above the 69.5 kDa predicted backbone | treat lysate with PNGase F and confirm the deglycosylated product shifts down toward ~69.5 kDa |
| Broad smear instead of sharp band | heterogeneous glycan occupancy and processing across the three N-glycosylation sites | run a longer gradient gel, extend transfer time for the higher-MW glycoprotein, or deglycosylate to sharpen the band |
| Multiple bands | the 9 annotated PRLR splice isoforms, including distinct membrane-bound and soluble forms | compare observed masses to isoform lengths or use isoform-selective antibodies to assign each band |
| No band in lysate | a soluble/secreted PRLR isoform released into the medium rather than retained in the cell pellet | analyze concentrated conditioned medium alongside whole-cell lysate |
| Weak or no signal | low endogenous PRLR expression in cell types with limited receptor density | enrich the membrane fraction, load more total protein, or use a tissue/cell line with known high PRLR expression |
| Fragments below expected size | proteolytic degradation of the glycosylated extracellular domain during lysis | prepare lysates fresh with protease inhibitors and compare against a mammalian-expressed positive control |
Experiment evidence
Western blot images showing PRLR band
PB9782 · PRLR
PA2087 · PRLR
PA2087 · PRLR
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PRLR Western blot
🧪For positive controls for PRLR in Western blot, you can use lysate from HEK293 cells transiently transfected with PRLR, since no HPA tissue or cell-line expression data is available to identify a confirmed endogenous positive source.
Positive control: PRLR-transfected HEK293 cells
Negative control: untransfected/mock-transfected HEK293 cells
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT).
⚠️Feasibility: With no HPA expression data to point to a validated endogenous tissue and PRLR being a single-pass membrane protein, a transfection/mock-transfection pair with membrane-optimized lysis is the most feasible control strategy rather than relying on native tissue lysates.
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 PRLR Western Blot Tips
Deeper troubleshooting and optimisation questions for PRLR, answered from its protein features.
Why does PRLR migrate above its 69.5 kDa predicted mass?
PRLR carries 3 N-glycosylation sites, and glycan attachment increases apparent molecular weight on SDS-PAGE. This accounts for the commonly observed 65-95 kDa smear or band instead of a sharp 69.5 kDa band. Glycosylation heterogeneity across cell types can also broaden the band.
Why does PRLR show a broad or diffuse band?
Variable occupancy at the 3 glycosylation sites produces a mix of glycoforms with slightly different masses, which run together as a smear rather than one sharp band. This is expected for a glycoprotein and not necessarily a specimen quality issue.
Which PRLR isoform does my antibody detect?
PRLR has 9 annotated isoforms from alternative splicing. Confirm the immunogen region falls within the isoform your antibody targets, since truncated or alternative-exon isoforms can appear as extra lower-molecular-weight bands or be missed entirely depending on epitope location.
Could a second band be a PRLR dimer?
PRLR homodimerizes upon prolactin binding. If samples are not fully reduced or heated, a disulfide-stabilized or hormone-induced dimer near double the monomer mass can appear. Use standard reducing, denaturing SDS-PAGE conditions with DTT or beta-mercaptoethanol to resolve the monomer cleanly.
How should blocking be optimized for PRLR detection?
Because PRLR is a glycoprotein with 2 disulfide bonds, avoid harsh reducing blocking conditions before antibody incubation and prefer BSA-based blocker if the antibody epitope is near a glycosylation or disulfide site, since milk glycoproteins can occasionally add background on glycoprotein targets.
What transfer method to use for PRLR Western blot?
PRLR is a single-pass type I membrane protein with glycosylation, so use wet transfer with reduced methanol (10 percent) and extended transfer time to move this larger, glycosylated species efficiently out of the gel onto PVDF.
How to quantify PRLR given multiple isoforms exist?
Quantify only the band matching your antibody's validated epitope and expected isoform mass, normalize to total protein rather than a single housekeeping band, and avoid summing unrelated bands from other isoforms into one signal.
What explains unexpected extra bands on a PRLR blot?
Extra bands can reflect distinct isoforms among the 9 splice variants, incompletely reduced disulfide-linked dimers, or partial glycosylation producing doublets. Compare band sizes against isoform-specific predicted masses and confirm reducing conditions before concluding nonspecific binding.
Boster reagents
Best PRLR Western Blot Antibodies
BosterBio's PRLR 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-Prolactin Receptor/PRLR Antibody Picoband®
Cat # PB9782
Real WB data
Anti-Prolactin Receptor/PRLR Antibody Picoband®
Cat # PA2087
Boster's anti-PRLR antibodies (PB9782, PA2087) are top-performing, extensively cited reagents validated by Western blot and orthogonally cross-checked against negative-tissue controls and complementary detection methods, ensuring specific, reproducible PRLR detection you can trust for critical experiments.
Which to pick: Both PB9782 and PA2087 include genuine Western blot validation images on PRLR-expressing samples, making either a reliable choice; since both are equally supported, selection can come down to lot availability or personal preference.
Source: BosterBio PRLR gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P16471.
- Human Protein Atlas. PRLR tissue expression.