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- Table of Contents
SMYD2 / N-lysine methyltransferase SMYD2 · Western blot design guide
Design a Western Blot for SMYD2
Source-linked SMYD2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SMYD2 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
SMYD2 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 | ~49.7 kDa | |
| Gel | 12–15% (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked SMYD2 Western Blot Protocol Options
The M04281 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| 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 | M04281; 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 SMYD2 Western Blot Band Size?
SMYD2 has a predicted mass of 49.7 kDa; isoforms may affect migration, but no empirical band position or distinct isoform bands are demonstrated.
| Band near 49.7 kDa | Consistent with the predicted SMYD2 mass; confirm identity with controls |
| Two bands at different positions | Could reflect isoforms 1 and 2, whose migration is not established |
| One band despite two isoforms | The isoforms may not resolve or may not both be detected |
| Band in nuclear or cytosolic fraction | Consistent with SMYD2 localization in both compartments |
💡Expected SMYD2 appearanceUniProt predicts SMYD2 at 49.7 kDa, but no empirical band size is supplied; confirm any candidate band with a positive control and an independent identity check.
| Predicted SMYD2 mass | Sets a 49.7 kDa reference, not a validated migration position |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Alternative splicing | May change isoform size, but distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SMYD2 detection in this sample is unconfirmed | Check a positive-control lysate and antibody performance |
| Band higher than expected | An isoform or unrelated signal is possible; identity is unconfirmed | Compare with a positive control and verify identity independently |
| Band lower than expected | An isoform or protein breakdown is possible; identity is unconfirmed | Use fresh lysate and verify identity independently |
| Multiple bands | Isoforms 1 and 2 are documented, but their band positions are unknown | Verify each band with an independent SMYD2 identity check |
| Weak or no signal | Signal may depend on the sampled cytosolic or nuclear fraction | Check both fractions and include a positive control |
| Fragments below expected size | Sample degradation is possible; no cleavage product is documented | Use fresh lysate and compare with an independent SMYD2 identity check |
Experiment evidence
Western blot images showing SMYD2 band
M04281 · SMYD2
A04281 · SMYD2
A04281 · SMYD2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SMYD2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SMYD2 in Western blot, you can use adrenal gland tissue, which has high 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: Adipose tissue is HPA-not-detected, but verify that it gives no SMYD2 signal in your lysate.
HPA tissue expression evidence for SMYD2
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 → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | 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 → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SMYD2 Western Blot Tips
Deeper troubleshooting and optimisation questions for SMYD2, answered from its protein features.
How should SMYD2 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 could SMYD2 isoforms affect the band pattern?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 273–433 of the canonical sequence and may produce a lower band if the antibody recognizes it. Check whether the antibody epitope lies in the retained sequence before assigning either band.
To detect both listed isoforms, select an epitope within canonical residues 1–272, which isoform 2 retains. An epitope within residues 273–433 would miss isoform 2 because that segment is absent. Check the antibody’s stated epitope before interpreting a missing lower band.
To detect both listed isoforms, select an epitope within canonical residues 1–272, which isoform 2 retains. An epitope within residues 273–433 would miss isoform 2 because that segment is absent. Check the antibody’s stated epitope before interpreting a missing lower band.
Does phosphorylation predict a visible SMYD2 band shift?
PTM · UniProt lists phosphoserine at canonical position 283. That site falls within the segment missing from isoform 2. Feature presence alone does not establish a visible shift; compare treated and untreated samples if testing a phosphorylation-dependent band pattern. Keep the canonical UniProt numbering explicit when comparing antibody or paper annotations.
Does this guide establish induction of SMYD2?
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 SMYD2?
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 M04281 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 multiple SMYD2 bands be quantified?
Quantitation · Define which band or bands the antibody detects before comparing samples. The two listed isoforms differ by deletion of canonical residues 273–433, so a combined signal and an isoform-specific signal answer different questions. Report the band positions used; no empirical SMYD2 band positions are supplied.
Where should SMYD2 appear relative to its predicted mass?
Interpretation · The canonical sequence has a predicted mass of 49.7 kDa. No observed band position is supplied, so use 49.7 kDa as a starting reference, not an expected apparent mass. The listed features alone do not explain a difference between predicted and observed migration.
UniProt lists SMYD2 in both the nucleus and cytosol. If using fractions, assess each compartment separately and account for fraction recovery when comparing amounts. A change in one fraction need not mean the total SMYD2 amount changed.
Isoform 2 lacks canonical residues 273–433 and is one possible explanation if the antibody recognizes its retained sequence. The supplied features do not identify an observed band, so position alone cannot establish its identity. Check epitope coverage and compare the pattern with an appropriate SMYD2-specific control.
UniProt lists SMYD2 in both the nucleus and cytosol. If using fractions, assess each compartment separately and account for fraction recovery when comparing amounts. A change in one fraction need not mean the total SMYD2 amount changed.
Isoform 2 lacks canonical residues 273–433 and is one possible explanation if the antibody recognizes its retained sequence. The supplied features do not identify an observed band, so position alone cannot establish its identity. Check epitope coverage and compare the pattern with an appropriate SMYD2-specific control.
Boster reagents
SMYD2 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-SMYD2 monoclonal Antibody
Cat # M04281
Real WB data
Anti-SMYD2/Kmt3C Antibody
Cat # A04281
The catalog reports two anti-SMYD2 antibodies with WB images. M04281 is a monoclonal antibody tested on 3T3, HT1080, A431 and Jurkat lysates at 1:1000. A04281 is a polyclonal antibody shown on unspecified cells at 1:2000. These examples do not establish universal species validation.
Which to pick: Both list human and mouse reactivity; A04281 also lists rat. Pick M04281 for a WB image with named cell lysates. A04281 has a WB image, but its caption says only ‘various cells’ and does not identify a rat sample.
Source: BosterBio SMYD2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NRG4.
- Human Protein Atlas. SMYD2 tissue expression.
- PMC9586978 — target-verified WB comparison
- PMC8755774 — target-verified WB comparison
- PMC12270048 — target-verified WB comparison