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- Table of Contents
DLX5 / Homeobox protein DLX-5 · Western blot design guide
Design a Western Blot for DLX5
Real validated DLX5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DLX5 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
DLX5 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 | ~31.5 kDa | |
| Observed band | ~35 kDa | |
| Gel | 10% (catalog M02523) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Fallopian tube (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
Real Curated DLX5 Western Blot Protocols
The M02523 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, human Hacat (catalog M02523) |
| Gel % | 10% (catalog M02523) |
| Load | 30 ug; reducing conditions (catalog M02523) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02523) |
| Membrane | nitrocellulose membrane (catalog M02523) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02523) |
| Primary antibody | M02523 · 1:500 (catalog M02523) |
| Primary incubation | overnight at 4°C (catalog M02523) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M02523) |
| Secondary incubation | 1.5 hour at RT (catalog M02523) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02523) |
| Detection | ECL (catalog M02523) |
Section 2
What Is the Expected DLX5 Western Blot Band Size?
DLX5 is predicted at 31.5 kDa and observed near 35 kDa; the cause of the difference is not established.
| Band near 35 kDa | Empirical DLX5 band reported in reducing whole-cell blots; confirm identity with controls |
| Band near 31.5 kDa | Near the predicted DLX5 sequence mass; identity still requires confirmation |
| Multiple bands | Isoforms 1 and 2 could contribute if their migration differs; distinct bands are not established |
| Weak band in whole-cell lysate | Nuclear DLX5 may be easier to detect after nuclear enrichment |
💡Expected DLX5 appearanceDLX5 has a predicted mass of 31.5 kDa and an empirical band near 35 kDa; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| Predicted DLX5 mass | 31.5 kDa from sequence; an empirical band appears near 35 kDa |
| Isoforms 1 and 2 | Alternative splicing may change size, but their relative masses and migration are unspecified |
| Serine 34 phosphorylation | MAPK14 phosphorylates this site in vitro; any effect on migration is unestablished |
| Serine 217 phosphorylation | MAPK14 phosphorylates this site in vitro; any effect on migration is unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear DLX5 may be scarce in the sampled whole-cell lysate | Check a nuclear-enriched fraction and a positive-control lysate |
| Band higher than expected | The reported 35 kDa band exceeds the 31.5 kDa prediction for an unestablished reason | Compare with the reported band and verify identity by DLX5 depletion |
| Band lower than expected | Alternative splicing could change size, but isoform migration is unknown | Verify identity by DLX5 depletion and compare isoform-specific detection if available |
| Multiple bands | Isoforms 1 and 2 may contribute, but distinct migration is unproven | Test which bands decrease after DLX5 depletion |
| Weak or no signal | Nuclear localization may limit DLX5 signal in whole-cell lysate | Check nuclear enrichment and a positive-control lysate |
Experiment evidence
Western blot images showing DLX5 band
M02523 · DLX5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DLX5 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for DLX5 in Western blot, you can use adrenal gland lysate.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Fallopian tube (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside DLX5.
⚠️Feasibility: HPA lists clear positive and negative tissues; DLX5 is nuclear, so nuclear-enriched lysate may aid detection.
HPA tissue expression evidence for DLX5
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 → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Fallopian tube | glandular cells | Not detected | Protein (IHC) | HPA → |
| Kidney | cells in glomeruli | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced DLX5 Western Blot Tips
Deeper troubleshooting and optimisation questions for DLX5, answered from its protein features.
How should DLX5 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 DLX5 isoform 2 affect band interpretation?
Isoforms · Isoform 2 replaces canonical residues 182–289 with NTALPCTRPA, making it substantially shorter. A lower band could therefore warrant isoform investigation, but the sequence difference alone does not establish where isoform 2 migrates on a blot.
An antibody recognizing canonical residues 182–289 could distinguish isoform 1 from isoform 2, which replaces that region. An antibody recognizing residues 1–181 would target sequence shared by both isoforms. Check the antibody’s documented epitope before assigning bands.
Using the supplied canonical UniProt coordinates, Ser34 lies in sequence retained by isoform 2. Canonical Ser217 lies within residues 182–289, which isoform 2 replaces. Specify the isoform and numbering convention when reporting either site.
An antibody recognizing canonical residues 182–289 could distinguish isoform 1 from isoform 2, which replaces that region. An antibody recognizing residues 1–181 would target sequence shared by both isoforms. Check the antibody’s documented epitope before assigning bands.
Using the supplied canonical UniProt coordinates, Ser34 lies in sequence retained by isoform 2. Canonical Ser217 lies within residues 182–289, which isoform 2 replaces. Specify the isoform and numbering convention when reporting either site.
Can phosphorylation explain an extra DLX5 band?
PTM · UniProt lists phosphoserine at positions 34 and 217, each produced by MAPK14 in vitro. These features do not show that phosphorylation occurs in the tested sample or causes a visible band shift. Do not assign an extra band to phosphorylation from position alone.
Does this guide establish induction of DLX5?
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 DLX5?
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 M02523 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 nuclear DLX5 be quantified across samples?
Quantitation · DLX5 is annotated as nuclear. If measuring it in nuclear extracts, prepare comparable fractions across samples and quantify the same identified band consistently. Account for the two isoforms when choosing which band to measure.
Why might DLX5 appear near 35 kDa instead of 31.5 kDa?
Interpretation · The observed band is about 35 kDa, versus a predicted mass of 31.5 kDa. The listed features do not establish the cause of that difference. Use the observed position as a reference, and assess additional bands separately.
First check whether the antibody recognizes the shared sequence or the region replaced in isoform 2. The two listed MAPK14 phosphorylation sites are in vitro findings and do not, by themselves, identify another band. Compare each candidate with the observed approximately 35 kDa band without assigning its cause solely from migration.
First check whether the antibody recognizes the shared sequence or the region replaced in isoform 2. The two listed MAPK14 phosphorylation sites are in vitro findings and do not, by themselves, identify another band. Compare each candidate with the observed approximately 35 kDa band without assigning its cause solely from migration.
Boster reagents
DLX5 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-Dlx5 Rabbit Monoclonal Antibody
Cat # M02523
M02523 is a rabbit monoclonal anti-DLX5 antibody listed for human, mouse, and rat. Its WB image shows a band near 35 kDa (expected 32 kDa) in four human cell lysates at 1:500. The supplied image does not show mouse or rat samples.
Which to pick: M02523 is the only listed option and has a WB image from HeLa, 293T, HaCaT, and RT4 whole cell lysates. For mouse or rat samples, the catalog lists reactivity, but the supplied WB image documents human samples only.
Source: BosterBio DLX5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P56178.
- Human Protein Atlas. DLX5 tissue expression.
- PMC6235898 — target-verified WB comparison
- PMC7522078 — target-verified WB comparison
- PMC6213991 — target-verified WB comparison