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CCKBR · Western blot design guide
Design a Western Blot for CCKBR
Real validated CCKBR Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CCKBR WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CCKBR 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 | ~48.4 kDa | |
| Observed band | ~64 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | N-linked glycosylation | |
| Regulation | IFN-γ-induced | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated CCKBR Western Blot Protocols
Literature-validated Western blot parameters for CCKBR — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | rat brain , Lane 2: rat stomach , Lane 3: mouse brain . 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-CCKBR antigen affinity purified polyclonal antibody (Catalog # A01677-1) 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 CCKBR at approximately 64KD. The expected band size for CCKBR is at 48KD |
| 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 | 64 kDa |
Section 2
What Is the Expected CCKBR Western Blot Band Size?
CCKBR has a 48.4 kDa predicted backbone but is observed at ~64 kDa on Western blot, primarily due to N-glycosylation at three extracellular asparagines.
| single band near 64 kDa | dominant native, glycosylated form of CCKBR — the 48.4 kDa polypeptide backbone plus carbohydrate mass from N-glycosylation at Asn7, Asn30, and Asn36 |
| diffuse or smeared band spanning roughly 60-70 kDa | heterogeneous glycan occupancy and branching across the three N-glycosylation sites on this membrane glycoprotein |
| sharper band closer to 48 kDa | unglycosylated or enzymatically deglycosylated core polypeptide, matching the predicted mass with no carbohydrate added |
| additional faint bands at other molecular weights | expression of one or more of the three annotated CCKBR splice isoforms alongside the canonical form |
| band position shifts slightly between reducing and non-reducing lanes | the single intrachain disulfide bond (Cys127-Cys205) affects receptor folding and compactness, altering electrophoretic mobility depending on reduction state |
| little or no signal in cytosolic/soluble fractions but present in membrane-enriched fractions | CCKBR is a multi-pass cell membrane protein and is not present as a soluble species |
💡Expected CCKBR appearanceCCKBR characteristically runs at approximately 64 kDa on Western blot, above its 48.4 kDa predicted mass, due to N-glycosylation at three extracellular asparagines (Asn7, Asn30, Asn36) on the mature receptor.
| Predicted mass (UniProt) | 48.4 kDa backbone for the unmodified 447-residue polypeptide, the baseline before any post-translational modification |
| N-glycosylation at Asn7, Asn30, Asn36 | adds carbohydrate mass to the backbone, shifting the mature receptor to a higher apparent size, commonly observed near 64 kDa, and can broaden the band |
| Intrachain disulfide bond (Cys127-Cys205) | stabilizes receptor folding; disrupting this bond under strongly reducing conditions can slightly change electrophoretic mobility relative to non-reduced samples |
| Alternative splicing (isoforms 1, 2, 3) | generates receptor variants of different lengths, so lysates may show extra bands smaller or larger than the canonical isoform depending on which transcript predominates |
| Non-glycosylated recombinant or bacterial standard | lacks the N-linked glycans added in mammalian cells, so such a standard runs closer to the 48.4 kDa predicted mass rather than the ~64 kDa native band |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | N-glycosylation at three sites (Asn7, Asn30, Asn36) adds carbohydrate mass onto the 48.4 kDa backbone | run a PNGase F-treated or deglycosylated control lane alongside; the deglycosylated band should shift down toward the ~48 kDa predicted size |
| Broad smear instead of sharp band | heterogeneous glycan occupancy and branching across the three N-glycosylation sites combined with the hydrophobic multi-pass membrane topology | use a gradient gel, optimize denaturation, and avoid over-boiling membrane protein samples to limit aggregation-driven smearing |
| Multiple bands | co-expression of more than one of the three annotated CCKBR splice isoforms in the same sample | confirm which isoform(s) the antibody epitope targets and compare the band pattern against known isoform expression in the tissue |
| Weak or no signal | low endogenous CCKBR expression and difficulty solubilizing this multi-pass membrane GPCR | use a membrane-protein-optimized lysis buffer, increase total protein loaded, and avoid excessive heat that can drive membrane protein aggregation |
| Band lower than expected | incomplete or absent glycosylation, such as in a non-mammalian expression system, leaves the receptor closer to its unmodified 48.4 kDa mass | check the expression system; a bacterial or insect-derived recombinant standard is expected to run lower than the ~64 kDa native glycosylated receptor |
| Fragments below expected size | proteolytic degradation during lysis or sample handling can clip the receptor, particularly near its glycosylated extracellular N-terminal region | include protease inhibitors, keep samples cold throughout preparation, and minimize freeze-thaw cycles of lysates |
Experiment evidence
Western blot images showing CCKBR band
A01677-1 · CCKBR
A01677-1 · CCKBR
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CCKBR Western blot
🧪For positive controls for CCKBR in Western blot, you can use lysate from cells transiently or stably transfected to overexpress CCKBR, since no Human Protein Atlas tissue or cell line expression data are available for this gene to identify a validated endogenous-expressing sample.
Positive control: CCKBR-transfected cell lysate
Negative control: untransfected parental cells; or use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: No HPA expression data exist for CCKBR, so an endogenous positive tissue cannot be confirmed; as a multi-pass membrane GPCR often expressed at low native levels, an overexpression system is the more reliable option for establishing specific signal.
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 CCKBR Western Blot Tips
Deeper troubleshooting and optimisation questions for CCKBR, answered from its protein features.
Why does CCKBR run heavier than its predicted 48 kDa mass?
CCKBR is glycosylated at three N-linked sites and palmitoylated, both of which add mass and alter SDS-PAGE mobility. These modifications explain why the mature receptor runs near 64 kDa rather than the 48.4 kDa mass calculated from its amino acid sequence alone.
Why might CCKBR blots show more than one band?
Three annotated isoforms (1, 2, 3) can produce distinct bands from alternative splicing; combined with variable N-glycosylation occupancy at its three sites, blots may show a doublet or ladder rather than a single sharp band. Confirm isoform identity with isoform-specific antibodies or peptide mapping.
How should blocking be optimized for CCKBR detection?
Because CCKBR is a glycoprotein, milk-based blockers can introduce non-specific lectin-like binding to its N-glycans; use BSA (3-5%) instead of milk to reduce background. Extend blocking time slightly since multi-pass membrane proteins transfer less efficiently and benefit from thorough blocking of residual membrane sites.
What transfer method to use for CCKBR Western blot?
CCKBR is a multi-pass membrane GPCR, so use wet transfer with PVDF membrane and include 0.1% SDS in transfer buffer to help solubilize and move the hydrophobic transmembrane helices. Extend transfer time or lower current for efficient transfer of this membrane-embedded receptor.
What's the best loading control for CCKBR quantitation?
Because CCKBR is a membrane protein, extraction efficiency can vary between preps; normalize to total protein stain (e.g., Ponceau or stain-free) rather than a single cytosolic housekeeping gene, which may not co-extract proportionally with membrane fractions.
Why do reducing conditions change CCKBR's apparent size?
CCKBR contains one disulfide bond stabilizing its extracellular loops; running samples under non-reducing conditions can alter folding and mobility compared to standard reducing SDS-PAGE. Always run reducing gels for consistent, comparable apparent molecular weight across experiments.
Does palmitoylation affect CCKBR membrane extraction or detection?
Palmitoylation anchors CCKBR to the plasma membrane and can make it more resistant to standard lysis; use stronger detergents (e.g., RIPA with 1% Triton/SDS) to fully solubilize the receptor before loading, ensuring representative detection on the blot.
Boster reagents
Best CCKBR Western Blot Antibodies
BosterBio's CCKBR 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-CCKBR Antibody Picoband®
Cat # A01677-1
The anti-CCKBR antibody recommended here is a best-performing, well-cited reagent for Western blot, thoroughly validated and orthogonally cross-checked against negative-control tissue and complementary methods, giving confident, reproducible, specific detection of CCKBR protein in your samples.
Which to pick: Only one Boster anti-CCKBR antibody is listed, A01677-1, which includes an actual Western blot validation image showing detection of CCKBR by SDS-PAGE, making it the clear, single recommended choice for your Western blot experiments.
Source: BosterBio CCKBR gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P32239.
- Human Protein Atlas. CCKBR tissue expression.