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PROK1 · Western blot design guide
Design a Western Blot for PROK1
Real validated PROK1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PROK1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
PROK1 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 | ~11.7 kDa | |
| Observed band | ~20 kDa | |
| Gel | 12–15% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Multiple disulfide bonds | |
| Regulation | Upregulated (immune signature) | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated PROK1 Western Blot Protocols
Literature-validated Western blot parameters for PROK1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | 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-Prokineticin 1 antigen affinity purified polyclonal antibody (Catalog # PB9881) 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 Prokineticin 1 at approximately 20 kDa. The expected band size for Prokineticin 1 is at 12 kDa |
| Gel % | 12–15% |
| 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 | 20 kDa |
Section 2
What Is the Expected PROK1 Western Blot Band Size?
PROK1 has an 11.7 kDa predicted precursor but its disulfide-stabilized secreted mature protein runs at approximately 20 kDa on SDS-PAGE due to a compact, SDS-resistant fold, not glycosylation.
| single band near 20 kDa | the empirical migration position of mature secreted PROK1, far above its calculated mass because its disulfide-stabilized fold resists normal SDS unfolding |
| band smaller than the 11.7 kDa full-length precursor mass | removal of the 19-residue signal peptide during secretion yields a mature chain lighter than the unprocessed precursor |
| little or no band in whole-cell lysate | PROK1 is a secreted protein, so it is efficiently exported and does not accumulate intracellularly |
| sharp band without smearing | PROK1 has no glycosylation sites, so no glycoform heterogeneity or sugar-driven smear is expected |
| shifted or additional band under non-reducing or partially reducing conditions | PROK1 carries five disulfide bonds; incomplete reduction leaves residual disulfide-constrained conformers that migrate differently |
| single band, no doublets | only one PROK1 isoform is reported, so no alternate-splice-driven second band is expected |
💡Expected PROK1 appearancePROK1 typically appears as a single band at approximately 20 kDa on reducing SDS-PAGE, well above its 11.7 kDa predicted mass because its five disulfide bonds hold the small secreted protein in a compact, slow-migrating fold.
| Predicted precursor mass (11.7 kDa, 105 aa) | sets the theoretical baseline size before any processing or folding effects are applied |
| Signal peptide cleavage (residues 1-19) | removes the N-terminal signal sequence during secretion, making the true mature chain lighter than the full-length precursor value |
| Five disulfide bonds (e.g. Cys26-Cys38, Cys32-Cys50, Cys37-Cys78, Cys60-Cys86, Cys80-Cys96) | lock the mature protein into a compact, SDS-resistant conformation that migrates markedly slower than its linear mass, producing the ~20 kDa apparent band |
| Absence of glycosylation sites | rules out glycan-driven mass increase or smearing, so the upward shift in apparent size is structural, not carbohydrate-based |
| Secreted subcellular localization | shifts detection toward conditioned media or biofluids, with correspondingly weaker signal expected in standard whole-cell lysates |
| Single reported isoform | no alternative splice forms are expected to appear as additional bands |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PROK1 is actively secreted and does not accumulate inside cells | probe conditioned media, serum, or plasma instead of, or in addition to, whole-cell lysate |
| Band higher than expected | the five disulfide bonds create a compact fold that resists complete SDS denaturation, and incomplete reduction compounds the effect | increase reducing agent concentration and boiling time to ensure full unfolding before comparing to the ~20 kDa reference band |
| Multiple bands | partial reduction of the five disulfide bonds yields a mixture of fully reduced and partially oxidized species with different mobilities | extend incubation with fresh DTT or beta-mercaptoethanol and re-run to collapse the mixture into a single species |
| Fragments below expected size | the small, disulfide-rich chain is susceptible to proteolytic clipping during sample handling | add protease inhibitors and keep samples cold throughout lysis and preparation |
| Weak or no signal | secreted PROK1 is present at low abundance in standard lysates relative to conditioned media or biofluids | concentrate the secreted fraction, load more protein, and use a high-sensitivity detection substrate |
| Broad smear instead of sharp band | residual, unevenly reduced disulfide bonds can produce a spread of closely spaced conformers rather than glycosylation-driven smearing | verify complete reduction and fresh reducing agent, since PROK1 has no glycosylation sites to explain smearing |
Experiment evidence
Western blot images showing PROK1 band
PB9881 · PROK1
PB9882 · PROK1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PROK1 Western blot
🧪For positive controls for PROK1 in Western blot, you can use conditioned medium or cell lysate from cells transiently overexpressing recombinant PROK1, since no HPA tissue expression data are available to identify a native positive source.
Positive control: PROK1-transfected cell lysate/medium
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: As a secreted protein with no HPA expression data to guide tissue selection, PROK1 is likely underrepresented in whole-cell lysates, so favor conditioned medium or an overexpression system and verify specificity with siRNA knockdown or a knockout line.
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 PROK1 Western Blot Tips
Deeper troubleshooting and optimisation questions for PROK1, answered from its protein features.
Why does PROK1 migrate near 20 kDa instead of predicted 11.7 kDa?
PROK1 contains five disulfide bonds that stabilize a compact tertiary fold, which can retard SDS-PAGE migration even after denaturation if reduction is incomplete. This compact, disulfide-stapled structure is a common reason the mature ~11.7 kDa secreted protein resolves closer to 20 kDa rather than at its calculated mass.
Are multiple isoforms expected to complicate PROK1 detection?
UniProt lists only one PROK1 isoform, so isoform-driven multi-banding is not expected. A single specific band should correspond to the mature secreted protein; any additional bands more likely reflect incomplete signal peptide processing, disulfide-linked aggregates, or reduction artifacts rather than true isoform variants.
Can disulfide bonds cause extra bands on non-reducing gels?
With five disulfide bonds, non-reducing SDS-PAGE can trap misfolded or disulfide-linked oligomeric species, producing bands that migrate faster or slower than the true monomer. Always run a fully reduced sample (DTT or beta-mercaptoethanol) alongside non-reduced samples to confirm the authentic monomeric band position.
What transfer method to use for PROK1 Western blot?
PROK1 is small (11.7 kDa predicted) and disulfide-stabilized, so use a lower-percentage or gradient gel with a low-molecular-weight-optimized run, then semi-dry or wet transfer at reduced voltage and shorter time. Use 0.2 micron PVDF or nitrocellulose to prevent blow-through of this small secreted protein.
How should blocking be optimized for this secreted mitogen?
As a small secreted growth factor lacking glycosylation or other modifications, PROK1 blots often benefit from BSA-based blocking rather than milk, since milk can contain endogenous growth factors that cross-react with antibodies. Try 5% BSA in TBST to reduce background while preserving low-abundance secreted signal.
How should PROK1 be quantified in conditioned media?
Because PROK1 is secreted, standard intracellular housekeeping controls like actin or GAPDH are inappropriate for conditioned media samples. Normalize instead to total secreted protein, cell number, or a co-secreted loading reference, and include recombinant PROK1 standards to enable relative quantitation across blots.
What explains an unexpected higher-MW band on PROK1 blots?
PROK1 has a cleavable signal peptide (residues 1-19); a higher-molecular-weight band may represent unprocessed pre-protein retained intracellularly rather than the mature secreted form. Disulfide-linked aggregates from incomplete reduction can also produce unexpected higher-MW species alongside the mature ~11.7 kDa (observed ~20 kDa) band.
Boster reagents
Best PROK1 Western Blot Antibodies
BosterBio's PROK1 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-Prokineticin 1/PROK1 Antibody Picoband®
Cat # PB9881
Real WB data
Anti-Prokineticin 1/PROK1 Antibody Picoband®
Cat # PB9882
Our recommended anti-PROK1 antibodies are top-performing, extensively cited, and rigorously validated through orthogonal approaches such as negative-tissue controls, delivering dependable, specific Western blot detection of Prokineticin 1 you can trust for consistent results.
Which to pick: Both PB9881 and PB9882 are validated with real Western blot images for Prokineticin 1 detection; either performs reliably, so selection can be based on availability, host species, or lot preference.
Source: BosterBio PROK1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P58294.
- Human Protein Atlas. PROK1 tissue expression.