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- Table of Contents
SEC63 / Translocation protein SEC63 homolog · Western blot design guide
Design a Western Blot for SEC63
Real validated SEC63 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SEC63 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
SEC63 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 | ~88 kDa | |
| Observed band | ~90 kDa | |
| Gel | 10% (catalog A04852-1) | |
| Positive control | Pancreas (IHC candidate; verify WB) +4 more | |
| Negative control | Oral mucosa (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 C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SEC63 Western Blot Protocols
The A04852-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A431, human 293T, human PC-3 (catalog A04852-1) |
| Gel % | 10% (catalog A04852-1) |
| Load | 30 ug; reducing conditions (catalog A04852-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04852-1) |
| Membrane | nitrocellulose membrane (catalog A04852-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04852-1) |
| Primary antibody | A04852-1 · 0.5 μg/mL (catalog A04852-1) |
| Primary incubation | overnight at 4°C (catalog A04852-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04852-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04852-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04852-1) |
| Detection | ECL (catalog A04852-1) |
Section 2
What Is the Expected SEC63 Western Blot Band Size?
SEC63 is predicted at 88 kDa and observed at approximately 90 kDa; the cause of the difference is not established.
| Band at approximately 90 kDa | Empirical SEC63 band in reducing whole-cell lysates; confirm identity with controls |
| Band near 88 kDa | Compatible with the predicted SEC63 mass; confirm identity with controls |
| Weak or absent band in lysate | ER membrane SEC63 may be poorly recovered during extraction |
| Close doublet near 90 kDa | Phosphorylation is annotated, but a visible migration difference is unproven |
💡Expected SEC63 appearanceSEC63 has a predicted mass of 88 kDa and an empirical band at approximately 90 kDa; the difference is unexplained, so confirm identity with appropriate controls.
| Predicted SEC63 mass | 88 kDa is the sequence-based reference mass |
| Phosphothreonine at residue 537 | A visible migration effect is not established |
| Phosphoserine at residue 742 | A visible migration effect is not established |
| Phosphoserine at residue 748 | A visible migration effect is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ER membrane SEC63 may be poorly extracted | Check membrane solubilization and a positive-control lysate |
| Band higher than expected | Identity of the higher band is uncertain | Compare with the approximately 90 kDa reference and use SEC63 knockdown |
| Band lower than expected | Identity of the lower band is uncertain | Check sample integrity and use SEC63 knockdown |
| Multiple bands | The listed phosphorylation sites do not establish distinct bands | Use SEC63 knockdown to identify the specific band |
| Weak or no signal | Membrane extraction or detection may be insufficient | Check membrane solubilization, loading, and a positive-control lysate |
Experiment evidence
Western blot images showing SEC63 band
A04852-1 · SEC63
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SEC63 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SEC63 in Western blot, you can use pancreas tissue, which HPA rates as high expression.
Positive control: Pancreas (IHC candidate; verify WB)
Negative control: Oral mucosa (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside SEC63.
⚠️Feasibility: As an ER membrane protein, SEC63 may require effective membrane protein extraction for a reliable signal.
HPA tissue expression evidence for SEC63
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 |
|---|---|---|---|---|
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | High | Protein (IHC) | HPA → |
| Skin | lymphocytes | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Cervix | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced SEC63 Western Blot Tips
Deeper troubleshooting and optimisation questions for SEC63, answered from its protein features.
How should SEC63 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 additional SEC63 bands represent isoforms?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign additional bands to SEC63 isoforms from these features alone; check their specificity with SEC63 depletion or another antibody.
Which SEC63 phosphorylation sites can a phospho-specific assay target?
PTM · The listed sites are phosphothreonine at UniProt position 537 and phosphoserine at UniProt positions 742 and 748. Check antibody numbering against the UniProt sequence before choosing a reagent, and validate site specificity. These features do not establish a visible band shift.
Measure phosphosite signal relative to total SEC63 from the same samples, keeping both signals within the assay’s linear range. Use consistent extraction because SEC63 is a multi-pass ER membrane protein. State whether the readout concerns UniProt Thr537, Ser742, or Ser748.
Measure phosphosite signal relative to total SEC63 from the same samples, keeping both signals within the assay’s linear range. Use consistent extraction because SEC63 is a multi-pass ER membrane protein. State whether the readout concerns UniProt Thr537, Ser742, or Ser748.
Does this guide establish induction of SEC63?
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 SEC63 Western blot?
Transfer · Choose a transfer setup that efficiently retains and transfers proteins around 88–90 kDa, then verify transfer with a total-protein stain. SEC63 is a multi-pass ER membrane protein, so optimize sample solubilization and transfer empirically; the features do not establish one preferred transfer method.
How should blocking be optimized for SEC63 detection?
Blocking · Compare blocking conditions with the chosen antibody and assess background around ~90 kDa. For a phosphosite antibody, check that the blocker preserves signal at the targeted UniProt site. The supplied features do not specify an optimal blocker.
How should SEC63 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.
Why does SEC63 run near 90 kDa instead of 88 kDa?
Interpretation · The observed ~90 kDa band is close to the predicted 88 kDa mass. SEC63 is a multi-pass ER membrane protein, so confirm band identity with a SEC63-specific control. Its listed phosphorylation sites alone do not explain the apparent mass difference.
Check band specificity before assigning an identity. The record lists one isoform, three phosphorylation sites, and no glycosylation sites; none alone establishes the cause of an extra band. SEC63 belongs to an ER translocon complex, but that annotation does not identify bands on a denaturing blot.
Check band specificity before assigning an identity. The record lists one isoform, three phosphorylation sites, and no glycosylation sites; none alone establishes the cause of an extra band. SEC63 belongs to an ER translocon complex, but that annotation does not identify bands on a denaturing blot.
Boster reagents
SEC63 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-SEC63 Antibody Picoband®
Cat # A04852-1
The catalog reports one anti-SEC63 antibody, A04852-1. Its Western blot image shows a band at approximately 90 kDa, matching the expected size, using human cell and rat and mouse tissue lysates. The supplied evidence is a product image; no publication validation is provided.
Which to pick: A04852-1 is the only listed option. It reports human, mouse, and rat reactivity and has a Western blot image using human A431, 293T, PC-3, and THP-1 cells, rat liver and kidney, and mouse liver. Use those tested samples to guide your choice.
Source: BosterBio SEC63 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UGP8.
- Human Protein Atlas. SEC63 tissue expression.
- PMC13267588 — target-verified WB comparison
- PMC2655295 — target-verified WB comparison
- PMC10150531 — target-verified WB comparison