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CREB3 / Cyclic AMP-responsive element-binding protein 3 · Western blot design guide
Design a Western Blot for CREB3
Source-linked CREB3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CREB3 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
CREB3 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~41.4 kDa | |
| Observed band | ~41–45 kDa | |
| Gel | 10% (catalog A03564-5) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Assay-dependent band sizes | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked CREB3 Western Blot Protocol Options
The A03564-5 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 A549, human U251, rat PC-12, mouse NIH/3T3 (catalog A03564-5) |
| Gel % | 10% (catalog A03564-5) |
| Load | 30 ug; reducing conditions (catalog A03564-5) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03564-5) |
| Membrane | nitrocellulose membrane (catalog A03564-5) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03564-5) |
| Primary antibody | A03564-5 · 0.5 μg/mL (catalog A03564-5) |
| Primary incubation | overnight at 4°C (catalog A03564-5) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03564-5) |
| Secondary incubation | 1.5 hour at RT (catalog A03564-5) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03564-5) |
| Detection | ECL (catalog A03564-5) |
Section 2
What Is the Expected CREB3 Western Blot Band Size?
CREB3 is predicted at 41.4 kDa and observed at ~41–45 kDa; the cause of any migration difference is not established.
| Band at ~41–45 kDa | Empirical CREB3 band near the 41.4 kDa predicted mass; confirm identity with antibody controls |
| Band above ~41–45 kDa | Could reflect modification at N-glycosylation sites Asn307 or Asn348; a visible shift is unproven |
| Band near twice the monomer size | Could represent retained CREB3 homodimer if it survives sample preparation |
| Several bands | Isoforms 1 and 2 are possible contributors; distinct migration is unproven |
💡Expected CREB3 appearanceCREB3 has a predicted mass of 41.4 kDa and an empirical band at ~41–45 kDa; confirm band identity with ordinary antibody controls.
| UniProt predicted mass | Places the reference monomer near 41.4 kDa |
| N-linked glycosylation site Asn307 | May affect apparent size; a visible shift is not established |
| N-linked glycosylation site Asn348 | May affect apparent size; a visible shift is not established |
| CREB3 homodimer | Could yield a band near twice the monomer size if retained during electrophoresis |
| Splice isoforms 1 and 2 | May differ in size, but their relative migration is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated CREB3 may be poorly recovered | Check a membrane-enriched fraction and extraction conditions |
| Band higher than expected | A retained homodimer or N-linked glycosylation is possible | Compare reducing preparation and glycosidase-treated samples with identity controls |
| Band lower than expected | Isoform identity or another cause is unresolved | Check antibody specificity and compare with an isoform-defined control |
| Broad smear instead of sharp band | Variable glycosylation at Asn307 or Asn348 is possible but unproven | Compare untreated and glycosidase-treated samples |
| Multiple bands | Isoforms 1 and 2 or retained homodimer may contribute | Compare isoform-defined controls and sample preparation conditions |
| Weak or no signal | Recovery of membrane-associated CREB3 may be low | Check membrane extraction and include a positive lysate control |
Experiment evidence
Western blot images showing CREB3 band
A03564-5 · CREB3
A03564-5 · CREB3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CREB3 Western blot
🧪For positive controls for CREB3 in Western blot, you can use an HPA-IHC candidate positive sample once one is identified; none is supplied here.
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 alongside the samples.
⚠️Feasibility: HPA provides no tissue data, so a positive tissue control cannot be selected from the supplied evidence.
HPA tissue expression evidence for CREB3
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 CREB3 Western Blot Tips
Deeper troubleshooting and optimisation questions for CREB3, answered from its protein features.
How should CREB3 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.
Could CREB3 isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Isoform 2 lacks residues 229–245 using UniProt numbering, so its sequence differs from isoform 1. This does not establish that the isoforms resolve as separate bands; check which isoforms the antibody recognizes.
What do the CREB3 glycosylation sites imply for band interpretation?
PTM · UniProt lists N-linked glycosylation at asparagines 307 and 348, using UniProt coordinates. These sites make glycosylation relevant when investigating band patterns, but their presence alone does not establish a visible shift or explain the observed 41–45 kDa range.
Does this guide establish induction of CREB3?
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.
How should transfer be checked for CREB3?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03564-5 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 CREB3 bands be quantified across samples?
Quantitation · Choose a band consistently across samples and report which band was measured. Two isoforms and two annotated N-linked glycosylation sites make it useful to assess whether multiple reproducible bands should be reported separately. The supplied features do not assign identities to individual bands.
How does the observed CREB3 band compare with its predicted mass?
Interpretation · CREB3 has a predicted mass of 41.4 kDa, and reported bands appear around 41–45 kDa. The supplied features do not establish why a band falls at a particular position within that range.
Isoform 1 is annotated as a single-pass type II protein in the endoplasmic reticulum membrane and is also reported at the Golgi apparatus. Include the membrane-containing material when preparing samples, and use the same preparation across comparisons.
Compare their positions with the reported ~41–45 kDa band, then consider the two annotated isoforms and N-linked sites at UniProt positions 307 and 348. Isoform 2 lacks UniProt residues 229–245. None of these features alone identifies an unexpected band or proves its cause.
Isoform 1 is annotated as a single-pass type II protein in the endoplasmic reticulum membrane and is also reported at the Golgi apparatus. Include the membrane-containing material when preparing samples, and use the same preparation across comparisons.
Compare their positions with the reported ~41–45 kDa band, then consider the two annotated isoforms and N-linked sites at UniProt positions 307 and 348. Isoform 2 lacks UniProt residues 229–245. None of these features alone identifies an unexpected band or proves its cause.
Boster reagents
CREB3 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-CREB3 Antibody Picoband®
Cat # A03564-5
The catalog reports one anti-CREB3 antibody, A03564-5, with a Western blot image showing an approximately 41 kDa band in human Caco-2, A549, and U251, rat PC-12, and mouse NIH/3T3 lysates. No publication evidence is supplied.
Which to pick: A03564-5 is the only listed option. Its WB image uses 30 μg whole-cell lysate per lane and 0.5 μg/mL primary antibody across the named human, rat, and mouse samples. Use those tested conditions as a starting point for your sample.
Source: BosterBio CREB3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O43889.
- Human Protein Atlas. CREB3 tissue expression.
- PMC12170202 — target-verified WB comparison
- PMC6814623 — target-verified WB comparison
- PMC10985101 — target-verified WB comparison