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GBF1 / Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 · Western blot design guide
Design a Western Blot for GBF1
Source-linked GBF1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GBF1 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
GBF1 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 | ~206.6 kDa | |
| Observed band | ~250 kDa | |
| Gel | 8% (catalog A01817-1) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Source-Linked GBF1 Western Blot Protocol Options
The A01817-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U251, human Caco-2, human A549, human HepG2, rat brain, mouse brain (catalog A01817-1) |
| Gel % | 8% (catalog A01817-1) |
| Load | 30 ug; reducing conditions (catalog A01817-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01817-1) |
| Membrane | nitrocellulose membrane (catalog A01817-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01817-1) |
| Primary antibody | A01817-1 · 0.5 μg/mL (catalog A01817-1) |
| Primary incubation | overnight at 4°C (catalog A01817-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01817-1) |
| Secondary incubation | 1.5 hour at RT (catalog A01817-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01817-1) |
| Detection | ECL (catalog A01817-1) |
Section 2
What Is the Expected GBF1 Western Blot Band Size?
GBF1 is predicted at 206.6 kDa but observed near 250 kDa on reducing Western blots; the cause of this difference is not established.
| Band near 250 kDa | Empirical GBF1 band in reducing whole-cell lysates; the reason it runs above the predicted mass is unestablished |
| Band near 206.6 kDa | Near GBF1's sequence-predicted mass; confirm identity with antibody controls |
| Several bands at different positions | GBF1 isoforms may contribute, but distinct migration has not been demonstrated |
| Band above the main band | A GBF1 oligomer is possible if it survives sample preparation; confirm its identity |
💡Expected GBF1 appearanceGBF1 has a predicted mass of 206.6 kDa, while antibody QC shows a band near 250 kDa in reducing whole-cell lysates; the cause of the difference is unestablished, so confirm identity with controls.
| UniProt predicted mass | 206.6 kDa is the sequence-based reference, while the observed band is near 250 kDa |
| Splice isoforms 1 and 4 | May differ in size; their relative masses and migration are not supplied |
| Splice isoforms 2 and 3 | May differ in size; their relative masses and migration are not supplied |
| GBF1 homodimer formation | Could yield a higher band if the complex survives sample preparation; no dimer band is established |
| Probable GBF1 homotetramer formation | Could yield a higher band if retained; no tetramer band is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | GBF1 may be poorly recovered from its Golgi-associated compartment | Check whole-cell extraction and use a GBF1-positive lysate control |
| Band higher than expected | The reported GBF1 band is near 250 kDa versus a predicted 206.6 kDa; the cause is unestablished | Compare with the reported positive lysates and confirm identity with an independent antibody or GBF1 depletion |
| Band lower than expected | A GBF1 isoform or proteolytic fragment is possible; no fragment mass is supplied | Check sample integrity and confirm the band by GBF1 depletion |
| Multiple bands | GBF1 has four named splice isoforms, though distinct band positions are unproven | Compare isoform-specific controls and verify candidate bands by GBF1 depletion |
| Weak or no signal | Recovery or transfer of this large Golgi-associated protein may be poor | Check extraction and high-molecular-weight transfer with a positive lysate |
| Fragments below expected size | GBF1 may undergo proteolysis during sample preparation | Prepare fresh lysate with protease inhibitors and compare with GBF1 depletion |
Experiment evidence
Western blot images showing GBF1 band
A01817-1 · GBF1
A01817-1 · GBF1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GBF1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for GBF1 in Western blot, you can use adrenal gland tissue, which has medium HPA staining.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: GBF1 is Golgi localized, and HPA identifies a not-detected tissue for a negative control.
HPA tissue expression evidence for GBF1
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GBF1 Western Blot Tips
Deeper troubleshooting and optimisation questions for GBF1, answered from its protein features.
How should GBF1 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 GBF1 isoforms produce separate bands?
Isoforms · Four isoforms are listed. Relative to canonical isoform 1, isoforms 3 and 4 change QQ to Q at UniProt positions 337–338; isoforms 2 and 3 lack positions 1495–1498. These short sequence differences do not establish that separate bands will be visible.
Which GBF1 phosphorylation sites matter when interpreting bands?
PTM · The supplied UniProt coordinates list phosphoserine at 350, 353, 663, 1299, 1319, 1321, 1336, 1476, 1774 and 1785; phosphothreonine at 508 and 1338; and phosphotyrosine at 1317. T1338 is attributed to AMPK. These are UniProt coordinates; paper or antibody numbering may differ. Site presence alone does not demonstrate a visible band shift.
UniProt identifies T1338 as phosphorylated by AMPK. If comparing conditions that alter AMPK activity, assess phosphorylation at that UniProt position separately from total GBF1. The feature does not establish that those conditions change total GBF1 abundance or band position.
UniProt identifies T1338 as phosphorylated by AMPK. If comparing conditions that alter AMPK activity, assess phosphorylation at that UniProt position separately from total GBF1. The feature does not establish that those conditions change total GBF1 abundance or band position.
Does this guide establish induction of GBF1?
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.
What transfer method to use for GBF1 Western blot?
Transfer · GBF1 is a large protein: 206.6 kDa predicted, with an observed band near 250 kDa. Choose and optimize a transfer method for high-mass proteins, then check transfer using a marker in that size range. The supplied features do not specify a transfer protocol.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01817-1 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 I quantify the GBF1 band?
Quantitation · Measure the consistently identified GBF1 band near the supplied observed position of 250 kDa using the same band boundaries across samples. Check any additional bands before including them in total GBF1 signal; the listed isoforms and phosphorylation sites do not establish which bands represent GBF1.
Why does GBF1 appear near 250 kDa instead of 206.6 kDa?
Interpretation · The supplied observed band is about 250 kDa, while the predicted mass is 206.6 kDa. The listed phosphorylation sites and isoforms do not, by themselves, explain that difference. Use the observed position as a guide, and verify band identity before assigning the difference to a specific modification.
Compare them with the 206.6 kDa predicted mass and the supplied observed band near 250 kDa. GBF1 has four listed isoforms and 13 phosphorylation sites, but those features alone cannot identify an unexpected band or prove a shift. Verify band identity before assigning it to an isoform or modification.
Compare them with the 206.6 kDa predicted mass and the supplied observed band near 250 kDa. GBF1 has four listed isoforms and 13 phosphorylation sites, but those features alone cannot identify an unexpected band or prove a shift. Verify band identity before assigning it to an isoform or modification.
Boster reagents
GBF1 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-GBF1 Antibody Picoband®
Cat # A01817-1
The catalog reports one anti-GBF1 WB antibody, A01817-1, with reported human, mouse, and rat reactivity. Its WB image shows human cell and rat and mouse brain lysates. The detected band is approximately 250 kDa, while the expected size is 207 kDa.
Which to pick: A01817-1 is the only listed option and has a WB image. Compare your sample with the reported human cell or rat and mouse brain lysates, and consider the difference between the observed 250 kDa band and expected 207 kDa size.
Source: BosterBio GBF1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q92538.
- Human Protein Atlas. GBF1 tissue expression.
- PMC6398479 — target-verified WB comparison