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SETD2 / Histone-lysine N-methyltransferase SETD2 · Western blot design guide
Design a Western Blot for SETD2
Real validated SETD2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SETD2 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
SETD2 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 | ~287.6 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| 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 + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated SETD2 Western Blot Protocols
The A00660 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of Mouse spleen, (catalog A00660) |
| Gel % | 4–12% gradient (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A00660) |
| Primary antibody | A00660 · 1:913 (catalog A00660) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A00660) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A00660) |
Section 2
What Is the Expected SETD2 Western Blot Band Size?
SETD2 is predicted at 287.6 kDa; isoforms and phosphorylation could affect migration, but no empirical band or feature-specific shift is established.
| Band near 287.6 kDa | consistent with predicted SETD2 size; confirm identity with an independent antibody or knockdown |
| Additional bands at different sizes | could reflect isoforms 1, 2, and 3; their migration is not established |
| Weak band in soluble lysate but stronger band in nuclear or chromatin extract | consistent with SETD2's nuclear and chromosome localization |
| Close doublet near the expected region | could reflect annotated phosphorylation; a visible shift is not established |
💡Expected SETD2 appearanceSETD2 has a predicted mass of 287.6 kDa, but no empirical band size is supplied; confirm any band with an independent antibody or knockdown.
| Predicted SETD2 mass | sets a 287.6 kDa sequence-based reference, not a measured migration |
| Splice isoform 1 | its individual mass and migration are not supplied |
| Splice isoform 2 | its size relative to the other isoforms is not supplied |
| Splice isoform 3 | its size relative to the other isoforms is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | nuclear or chromosome-associated SETD2 may be poorly recovered | check nuclear or chromatin extraction and include a fractionation control |
| Band higher than expected | identity or apparent migration is uncertain without an empirical SETD2 band | verify with an independent antibody or SETD2 knockdown |
| Band lower than expected | a splice isoform or sample degradation may contribute | compare antibody epitopes and test band loss after SETD2 knockdown |
| Multiple bands | annotated isoforms or phosphorylation could contribute, but their migration is unknown | check band identity by knockdown and assess phosphorylation with phosphatase treatment |
| Weak or no signal | SETD2 may be underrepresented after soluble extraction | check nuclear or chromatin recovery and sample loading |
| Fragments below expected size | sample degradation may generate smaller species | repeat with fresh lysate and protease inhibitors, then verify SETD2 identity |
Experiment evidence
Western blot images showing SETD2 band
A00660 · SETD2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SETD2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SETD2 in Western blot, you can use adrenal gland tissue, which shows medium HPA expression.
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: SETD2 is nuclear, so nuclear lysates may give a clearer signal than whole-cell lysates.
HPA tissue expression evidence for SETD2
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 → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | glandular 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 → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SETD2 Western Blot Tips
Deeper troubleshooting and optimisation questions for SETD2, answered from its protein features.
How should SETD2 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 SETD2 isoforms produce smaller bands?
Isoforms · Yes. UniProt lists three isoforms: isoform 3 lacks canonical residues 1573–2564, and isoform 2 lacks 1715–2564. Both could yield bands smaller than full-length SETD2. Check whether the antibody's epitope lies in a retained region before assigning a band to either isoform.
Decide whether the measurement targets canonical SETD2 or all detectable isoforms. Isoforms 2 and 3 lack distinct C-terminal stretches, so first check which isoforms the antibody can recognize. Quantify consistently defined bands separately if they resolve; do not assume every SETD2-reactive band represents the same isoform.
Decide whether the measurement targets canonical SETD2 or all detectable isoforms. Isoforms 2 and 3 lack distinct C-terminal stretches, so first check which isoforms the antibody can recognize. Quantify consistently defined bands separately if they resolve; do not assume every SETD2-reactive band represents the same isoform.
Can phosphorylation explain a shifted SETD2 band?
PTM · UniProt lists 30 modified residues, including phosphoserines at positions 1413, 1415 and 1417 and phosphothreonines at 1853 and 1872. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible shift or explain a difference from 287.6 kDa.
Does this guide establish induction of SETD2?
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 SETD2 Western blot?
Transfer · SETD2's predicted mass is 287.6 kDa, so choose and optimize a transfer setup for a high-molecular-weight protein. Check transfer completeness near that size before interpreting a weak or absent band; the supplied features do not identify one required transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00660 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 SETD2 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.
Where should full-length SETD2 migrate?
Interpretation · The canonical 2,564-residue sequence has a predicted mass of 287.6 kDa. Use this as a reference when identifying a high-molecular-weight band; no observed band position is supplied, and the listed features do not establish an apparent-mass shift.
Compare them with the documented isoform deletions: residues 1715–2564 are missing in isoform 2, and 1573–2564 in isoform 3. An antibody epitope within a deleted stretch would argue against the corresponding isoform assignment. Band size alone cannot establish identity.
UniProt places SETD2 in the nucleus and on chromosomes. When comparing preparations, account for whether nuclear or chromosomal material was recovered; differences in recovery could affect band intensity independently of SETD2 abundance.
Compare them with the documented isoform deletions: residues 1715–2564 are missing in isoform 2, and 1573–2564 in isoform 3. An antibody epitope within a deleted stretch would argue against the corresponding isoform assignment. Band size alone cannot establish identity.
UniProt places SETD2 in the nucleus and on chromosomes. When comparing preparations, account for whether nuclear or chromosomal material was recovered; differences in recovery could affect band intensity independently of SETD2 abundance.
Boster reagents
SETD2 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-SETD2 Antibody
Cat # A00660
A00660 is an anti-SETD2 antibody listed as reactive with human, mouse, and rat. Its Western blot image uses mouse spleen extract (25 µg per lane) at 1:913 dilution. The supplied image documents this sample and condition only.
Which to pick: A00660 is the only listed option and has a Western blot image from mouse spleen. Its listed reactivity includes human, mouse, and rat, but the supplied blot shows only mouse spleen.
Source: BosterBio SETD2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BYW2.
- Human Protein Atlas. SETD2 tissue expression.
- PMC10690240 — target-verified WB comparison
- PMC6985262 — target-verified WB comparison
- PMC9477841 — target-verified WB comparison