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- Table of Contents
GSPT1 / Eukaryotic peptide chain release factor GTP-binding subunit ERF3A · Western blot design guide
Design a Western Blot for GSPT1
Source-linked GSPT1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GSPT1 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
GSPT1 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 | ~55.8 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (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 | — | |
| Caveat | — | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked GSPT1 Western Blot Protocol Options
The A07761 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | 3T3 cells (catalog A07761) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A07761; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected GSPT1 Western Blot Band Size?
GSPT1 has a predicted mass of 55.8 kDa; three annotated isoforms could affect migration, but no distinct band pattern is demonstrated.
| Band near 55.8 kDa | Consistent with the predicted GSPT1 mass; confirm identity with controls |
| Several discrete bands | Could reflect isoforms 1, 2, and 3; their migration is not established |
| Band above 55.8 kDa | An isoform is possible, but the band identity needs confirmation |
| Band below 55.8 kDa | An isoform is possible, but the band identity needs confirmation |
💡Expected GSPT1 appearanceUniProt predicts 55.8 kDa for GSPT1; no empirical band size or isoform migration is supplied, so confirm any candidate band with ordinary identity controls.
| UniProt predicted mass | Sets a 55.8 kDa reference for GSPT1 |
| Splice isoform 1 | Its individual apparent mass is not supplied |
| Splice isoform 2 | Its apparent size may differ; no individual mass is supplied |
| Splice isoform 3 | Its apparent size may differ; no individual mass is supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Multiple bands | Isoforms 1, 2, and 3 are annotated, but distinct bands are unproven | Check isoform coverage and confirm bands with GSPT1 depletion |
| Band higher than expected | Its identity and migration cause are unestablished | Compare with GSPT1 depletion and an independent antibody |
| Band lower than expected | An alternative isoform is possible; its size is unknown | Check antibody epitope coverage and confirm with GSPT1 depletion |
| No band in lysate | GSPT1 may be below detection or the blot may have failed | Check loading and transfer, then use a positive control lysate |
| Weak or no signal | Target abundance or antibody recognition may be insufficient | Increase lysate input and verify the antibody with a positive control |
Experiment evidence
Western blot images showing GSPT1 band
A07761 · GSPT1
A07761-1 · GSPT1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GSPT1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for GSPT1 in Western blot, you can use appendix tissue, which has high HPA expression.
Positive control: Appendix (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: Adipose tissue is a feasible negative control because HPA reports GSPT1 as not detected there.
HPA tissue expression evidence for GSPT1
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 |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | 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 → |
| Caudate | neuronal cells | Low | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Low | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Low | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced GSPT1 Western Blot Tips
Deeper troubleshooting and optimisation questions for GSPT1, answered from its protein features.
Where should the main GSPT1 band appear?
Band shift · The canonical sequence is 499 residues with a predicted mass of 55.8 kDa. Use this as a reference, not a guaranteed migration position; no observed band size is supplied.
Can GSPT1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1, 2, and 3 with alternative N-terminal sequences. They could migrate differently, but these features alone do not establish that separate bands will be visible.
In UniProt coordinates, the alternative sequence annotated at position 1 replaces the initial methionine in isoforms 2 and 3; an additional sequence is annotated as missing at position 8 in isoform 2. Check which sequence your antibody recognizes before assigning a band to an isoform.
Decide whether the measurement should include all three listed isoforms or distinguish them. Check the antibody’s recognized sequence against the annotated N-terminal differences, and quantify bands consistently with that choice.
In UniProt coordinates, the alternative sequence annotated at position 1 replaces the initial methionine in isoforms 2 and 3; an additional sequence is annotated as missing at position 8 in isoform 2. Check which sequence your antibody recognizes before assigning a band to an isoform.
Decide whether the measurement should include all three listed isoforms or distinguish them. Check the antibody’s recognized sequence against the annotated N-terminal differences, and quantify bands consistently with that choice.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of GSPT1?
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 GSPT1?
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 A07761 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 GSPT1 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Does the predicted mass explain a shifted GSPT1 band?
Interpretation · The predicted mass is 55.8 kDa, and alternative sequences are annotated, but no empirical band position is supplied. Compare each observed band with the relevant isoform sequence; the listed features alone cannot explain a specific mass difference.
GSPT1 is listed as part of an eRF1–eRF3–GTP ternary complex and a transient SURF complex. Those annotations do not establish that a complex survives Western blot preparation or produces an extra band. Check band identity before assigning it to a complex.
GSPT1 is listed as part of an eRF1–eRF3–GTP ternary complex and a transient SURF complex. Those annotations do not establish that a complex survives Western blot preparation or produces an extra band. Check band identity before assigning it to a complex.
Boster reagents
GSPT1 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-eRF3a GSPT1 Antibody
Cat # A07761
Real WB data
Anti-eRF3a (D128) GSPT1 Antibody
Cat # A07761-1
Both anti-GSPT1 antibodies list Human, Mouse, and Rat reactivity and have WB images. A07761 shows 3T3 cells; A07761-1 shows SGC7901, Panc1, PC3, AML-12, and PC12 lysates at 1:500. These examples do not establish validation across all listed species.
Which to pick: Choose based on the reported sample: A07761 has a 3T3 WB example, while A07761-1 has examples from five cell lysates at 1:500. Both list the same species reactivity; neither has a unique reactivity advantage in the supplied evidence.
Source: BosterBio GSPT1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P15170.
- Human Protein Atlas. GSPT1 tissue expression.
- PMC8165035 — target-verified WB comparison
- PMC12186427 — target-verified WB comparison
- PMC12354682 — target-verified WB comparison