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- Table of Contents
SET / Protein SET · Western blot design guide
Design a Western Blot for SET
Real validated SET Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SET 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
SET 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 | ~33.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Alternative isoform sizes | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated SET Western Blot Protocols
The M02211 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HepG2 cell lysate (catalog M02211) |
| 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 | M02211; 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 SET Western Blot Band Size?
SET has a predicted 33.5 kDa monomer; splice isoforms may add bands, and its homodimer may appear higher if it remains associated.
| Band near 33.5 kDa | SET monomer near its predicted mass |
| Several bands at different positions | SET isoforms 1, 2, 3, and 4 may differ in size |
| Band near twice the monomer size under nonreducing conditions | SET homodimer may remain associated |
| Fragments below the main band | SET may have undergone GZMA cleavage |
💡Expected SET appearanceExpect a SET monomer near its predicted 33.5 kDa, with possible additional bands from isoforms 1–4 or a higher band if the homodimer remains associated.
| Predicted SET mass | Places the full-length monomer near 33.5 kDa |
| Isoforms 1 and 2 | Alternative splicing may produce bands at different positions |
| Isoforms 3 and 4 | Alternative splicing may produce additional bands at different positions |
| SET homodimer | May produce a band near twice the monomer size if it remains associated |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SET may be below detection in the sampled lysate | Check sample loading and enrich the cytosolic or nuclear fraction |
| Band higher than expected | SET homodimer may remain associated | Compare denaturing and nonreducing preparations |
| Band lower than expected | A different SET splice isoform may be detected | Check which isoforms the antibody recognizes |
| Multiple bands | SET has four named isoforms | Compare bands with isoform-specific expression or depletion |
| Weak or no signal | SET abundance may differ between sampled compartments | Compare cytosolic and nuclear fractions |
| Fragments below expected size | GZMA cleavage of SET may generate fragments | Check whether the sample underwent GZMA exposure |
Experiment evidence
Western blot images showing SET band
M02211 · SET
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SET Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SET in Western blot, you can use adrenal gland lysate, which shows high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside SET.
⚠️Feasibility: No HPA tissue is listed as not detected, so use siRNA knockdown or a KO line for a reliable negative control.
HPA tissue expression evidence for SET
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 | High | Protein (IHC) | HPA → |
| 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 → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Medium | Protein (IHC) | HPA → |
| Lung | alveolar cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced SET Western Blot Tips
Deeper troubleshooting and optimisation questions for SET, answered from its protein features.
How should SET 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.
How can SET isoforms affect band interpretation?
Isoforms · UniProt lists four SET isoforms. Check which isoforms the antibody recognizes before assigning nearby bands; the supplied features do not give isoform-specific masses.
Could SET modifications explain multiple bands?
PTM · SET has eight annotated modified residues and keywords for acetylation, methylation, phosphorylation, and ubiquitin-like conjugation. Compare bands under conditions relevant to the modification of interest, but do not assign a band to a specific modification from size alone.
Does this guide establish induction of SET?
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 SET?
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 M02211 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 SET 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 might SET migrate differently from its predicted 33.5 kDa mass?
Interpretation · SET has four isoforms and eight annotated modified residues. These features can affect the bands detected, but no observed band size is supplied. Use 33.5 kDa as a reference, not a confirmed migration position.
SET is listed in the cytosol, associated with the endoplasmic reticulum, and in the nucleoplasm. Use consistent fractionation when comparing samples, and interpret changes in one fraction alongside the others.
The supplied location note says SET moves rapidly to the nucleus following CTL attack and GZMA cleavage. If comparing attacked and untreated cells, examine nuclear and cytoplasmic fractions and consider cleavage when interpreting changed bands.
Consider the four isoforms, annotated modifications, and GZMA-associated cleavage before assigning an unexpected band. The supplied record gives no empirical band size, so confirm band identity with an independent specificity check.
SET is listed in the cytosol, associated with the endoplasmic reticulum, and in the nucleoplasm. Use consistent fractionation when comparing samples, and interpret changes in one fraction alongside the others.
The supplied location note says SET moves rapidly to the nucleus following CTL attack and GZMA cleavage. If comparing attacked and untreated cells, examine nuclear and cytoplasmic fractions and consider cleavage when interpreting changed bands.
Consider the four isoforms, annotated modifications, and GZMA-associated cleavage before assigning an unexpected band. The supplied record gives no empirical band size, so confirm band identity with an independent specificity check.
Boster reagents
SET 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-SET Monoclonal Antibody
Cat # M02211
M02211 is the listed anti-SET antibody with a Western blot image from HepG2 cell lysate. The supplied record does not establish best-performing status, citation history, thorough validation, or orthogonal cross-validation against negative tissue or complementary methods.
Which to pick: Only M02211 is listed. Choose it if a HepG2 lysate Western blot example fits your experiment; confirm reactivity and validation for your sample before use.
Source: BosterBio SET gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q01105.
- Human Protein Atlas. SET tissue expression.
- PMC6063425 — target-verified WB comparison
- PMC6949222 — target-verified WB comparison