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- Table of Contents
SIM2 / Single-minded homolog 2 · Western blot design guide
Design a Western Blot for SIM2
Source-linked SIM2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SIM2 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
SIM2 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 | ~73.2 kDa | |
| Observed band | ~65 kDa | |
| Gel | 5–20% (catalog A04436-2) | |
| Positive control | Bone marrow (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 | — | |
| Caveat | Band below predicted size | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked SIM2 Western Blot Protocol Options
The A04436-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human PC-3, human HepG2, monkey COS-7, rat skeletal muscle, rat brain, mouse skeletal muscle, mouse brain (catalog A04436-2) |
| Gel % | 5–20% (catalog A04436-2) |
| Load | 30 ug; reducing conditions (catalog A04436-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04436-2) |
| Membrane | nitrocellulose membrane (catalog A04436-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04436-2) |
| Primary antibody | A04436-2 · 0.5 μg/mL (catalog A04436-2) |
| Primary incubation | overnight at 4°C (catalog A04436-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04436-2) |
| Secondary incubation | 1.5 hour at RT (catalog A04436-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04436-2) |
| Detection | ECL (catalog A04436-2) |
Section 2
What Is the Expected SIM2 Western Blot Band Size?
SIM2 is predicted at 73.2 kDa, while antibody QC reports ~65 kDa; the cause of that difference is not established.
| Band near 65 kDa | Empirical SIM2 band; confirm identity with a specific control |
| Band near 73.2 kDa | Near the predicted SIM2 mass; identity still needs confirmation |
| Multiple bands near the SIM2 region | SIM2 and SIM2S are possible contributors, but distinct migration is unproven |
| Weak or absent band in a cytosolic fraction | SIM2 is nuclear, so the fraction may contain little target |
💡Expected SIM2 appearanceSIM2 has a predicted mass of 73.2 kDa, while an antibody QC blot reports a band near 65 kDa; the cause of the difference is unestablished, so confirm identity with appropriate controls.
| 73.2 kDa predicted mass | Provides a sequence-based reference; the reported band migrates near 65 kDa |
| SIM2 splice isoform | Its mass relative to SIM2S is not supplied |
| SIM2S splice isoform | Its mass relative to SIM2 is not supplied |
| Alternative splicing of SIM2 and SIM2S | Could affect apparent size, but distinct band positions are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear SIM2 may be poorly recovered or scarce in the sample | Check nuclear extraction and use a positive control |
| Band higher than expected | The band’s identity or migration is unresolved | Compare with a positive control and verify target specificity |
| Band lower than expected | The reported 65 kDa band is below the 73.2 kDa prediction; the cause is unestablished | Verify identity with SIM2 depletion or another antibody |
| Multiple bands | SIM2 and SIM2S are possible contributors, but their migration is unknown | Test band identity with isoform-specific reagents or SIM2 depletion |
| Weak or no signal | Low target abundance or poor recovery of nuclear protein | Check sample loading and nuclear enrichment with controls |
Experiment evidence
Western blot images showing SIM2 band
A04436-2 · SIM2
A04436 · SIM2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SIM2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SIM2 in Western blot, you can use bone marrow lysate, which HPA rates highly positive.
Positive control: Bone marrow (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA lists only low-expression negative candidates, so use siRNA knockdown or a KO line for a clear negative control.
HPA tissue expression evidence for SIM2
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial 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 → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Prostate | glandular cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced SIM2 Western Blot Tips
Deeper troubleshooting and optimisation questions for SIM2, answered from its protein features.
How should SIM2 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.
Which SIM2 regions distinguish the two isoforms?
Isoforms · In UniProt canonical numbering, residues 526–570 are replaced in SIM2S, and residues 571–667 are absent. An antibody against the canonical 571–667 region should distinguish canonical SIM2 from SIM2S if its epitope is accessible. Check the antibody’s stated epitope and numbering convention.
Use an antibody whose epitope lies in sequence shared by SIM2 and SIM2S, then assess whether distinct bands can be resolved. A shared epitope does not guarantee equal antibody response or establish band identity; use isoform specific confirmation before assigning or comparing signals.
Use an antibody whose epitope lies in sequence shared by SIM2 and SIM2S, then assess whether distinct bands can be resolved. A shared epitope does not guarantee equal antibody response or establish band identity; use isoform specific confirmation before assigning or comparing signals.
Do UniProt features support a modification based SIM2 band shift?
PTM · The supplied UniProt record lists no modified residues or glycosylation sites. It therefore provides no specific modification to assign to a shifted band. Compare apparent masses and isoform sensitive antibody results, but do not attribute the observed 65 kDa band to a modification from this record.
Does this guide establish induction of SIM2?
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 SIM2?
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 A04436-2 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.
Should SIM2 quantitation use a nuclear fraction?
Quantitation · UniProt places SIM2 in the nucleus. A nuclear fraction is therefore a relevant sample for measuring SIM2, provided fraction preparation and loading are consistent across samples. State whether measurements use nuclear extracts or whole cell lysates when comparing results.
Why might SIM2 appear near 65 kDa instead of 73.2 kDa?
Interpretation · The canonical SIM2 sequence has a predicted mass of 73.2 kDa, while the supplied Western blot observation is about 65 kDa. SIM2 has a shorter splice isoform, SIM2S, but these features alone do not establish which isoform produced the band or explain its migration.
Possibly. SIM2S replaces canonical residues 526–570 with a different sequence and lacks residues 571–667, making it shorter than canonical SIM2. The supplied features do not give an observed mass for SIM2S, so confirm the band’s identity with an isoform specific reagent or another identification method.
First consider the documented splice variants: SIM2S has a changed sequence at canonical positions 526–570 and lacks 571–667. Check whether each band reacts with antibodies against shared and canonical specific regions. SIM2 also heterodimerizes with ARNT, but that feature alone does not establish the identity of a higher band on a Western blot.
Possibly. SIM2S replaces canonical residues 526–570 with a different sequence and lacks residues 571–667, making it shorter than canonical SIM2. The supplied features do not give an observed mass for SIM2S, so confirm the band’s identity with an isoform specific reagent or another identification method.
First consider the documented splice variants: SIM2S has a changed sequence at canonical positions 526–570 and lacks 571–667. Check whether each band reacts with antibodies against shared and canonical specific regions. SIM2 also heterodimerizes with ARNT, but that feature alone does not establish the identity of a higher band on a Western blot.
Boster reagents
SIM2 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-SIM2 Antibody Picoband®
Cat # A04436-2
Real WB data
Anti-Single-minded homolog 2 SIM2 Antibody
Cat # A04436
Two anti-SIM2 antibodies have WB images. A04436-2 shows a band near 65 kDa, versus an expected 73 kDa, in named human, monkey, rat, and mouse samples. A04436 lists human reactivity, but its image caption does not identify the cell lines.
Which to pick: For the specific human cell lines or monkey, rat, and mouse tissues named in its caption, consider A04436-2. For other human samples, compare both antibodies; A04436’s WB caption does not identify its cell lines. Check the 65 versus 73 kDa discrepancy when interpreting A04436-2.
Source: BosterBio SIM2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14190.
- Human Protein Atlas. SIM2 tissue expression.
- PMC10826595 — target-verified WB comparison
- PMC5891185 — target-verified WB comparison
- PMC3235151 — target-verified WB comparison