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- Table of Contents
LIG4 / DNA ligase 4 · Western blot design guide
Design a Western Blot for LIG4
Real validated LIG4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LIG4 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
LIG4 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 | ~104 kDa | |
| Observed band | ~104 kDa | |
| Gel | 8% (catalog A01685-3) | |
| Positive control | Gallbladder (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 | XRCC4-dependent stability | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated LIG4 Western Blot Protocols
The A01685-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Ramos, human HepG2, human jurkat (catalog A01685-3) |
| Gel % | 8% (catalog A01685-3) |
| Load | 30 ug; reducing conditions (catalog A01685-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01685-3) |
| Membrane | nitrocellulose membrane (catalog A01685-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01685-3) |
| Primary antibody | A01685-3 · 0.5 μg/mL (catalog A01685-3) |
| Primary incubation | overnight at 4°C (catalog A01685-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01685-3) |
| Secondary incubation | 1.5 hour at RT (catalog A01685-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01685-3) |
| Detection | ECL (catalog A01685-3) |
Section 2
What Is the Expected LIG4 Western Blot Band Size?
LIG4 is predicted at 104 kDa and observed at ~104 kDa; no difference in migration is established.
| Band at ~104 kDa | Matches the predicted mass and observed LIG4 band |
| Single sharp band near 104 kDa | Consistent with the one listed LIG4 isoform |
| ~104 kDa band enriched in nuclear fraction | Consistent with LIG4's nuclear location |
| Weak ~104 kDa band | Could reflect reduced LIG4 stability when XRCC4 is deficient |
💡Expected LIG4 appearanceLIG4 is predicted at 104 kDa and observed at ~104 kDa; confirm band identity with LIG4 depletion or another appropriate antibody control.
| UniProt predicted mass | Places the expected band near 104 kDa |
| UniProt molecular weight of 103971 Da | Rounds to approximately 104 kDa |
| Predicted full-length size | Provides the 104 kDa reference for interpreting bands |
| Predicted LIG4 size | Agrees with the observed ~104 kDa band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear LIG4 may be poorly recovered | Check nuclear extraction with a nuclear marker |
| Band higher than expected | No supplied feature establishes a larger LIG4 band | Test band identity by LIG4 depletion |
| Band lower than expected | No supplied feature establishes a smaller LIG4 band | Test band identity by LIG4 depletion |
| Multiple bands | Only one LIG4 isoform is listed | Identify the ~104 kDa band by LIG4 depletion |
| Weak or no signal | LIG4 stability depends on XRCC4 | Check XRCC4 status and nuclear protein recovery |
Experiment evidence
Western blot images showing LIG4 band
A01685-3 · LIG4
M01685 · LIG4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for LIG4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for LIG4 in Western blot, you can use gallbladder tissue, which has high HPA expression.
Positive control: Gallbladder (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 the samples.
⚠️Feasibility: HPA lists no not-detected tissue, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for LIG4
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 |
|---|---|---|---|---|
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Placenta | decidual cells | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | 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 |
|---|---|---|---|---|
| Esophagus | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Low | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced LIG4 Western Blot Tips
Deeper troubleshooting and optimisation questions for LIG4, answered from its protein features.
How should LIG4 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 LIG4 isoforms explain multiple bands?
Isoforms · Only one isoform is listed, with no alternative sequence supplied. Do not assign additional bands to isoforms from these features alone.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of LIG4?
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 LIG4 Western blot?
Transfer · LIG4 is approximately 104 kDa. Check transfer of proteins near that size on the membrane and post-transfer gel when optimizing your method; these features do not specify a particular transfer setup.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01685-3 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 XRCC4 affect LIG4 quantitation?
Quantitation · XRCC4 is required for LIG4 stability. If XRCC4 differs between samples, a change in LIG4 signal may reflect altered stability; account for that when interpreting LIG4 abundance.
Should LIG4 migrate above its predicted mass?
Interpretation · LIG4 is predicted at 104 kDa, and the reported band is approximately 104 kDa. The supplied features do not establish a reason to expect a shift.
LIG4 is listed as nuclear. Check the nuclear fraction when comparing its signal across fractionated samples.
LIG4 is associated with DNA damage and repair, but the supplied features do not establish that damage treatment increases its abundance. Measure treated and control samples before inferring induction.
Compare them with the approximately 104-kDa expected band. The supplied record lists one isoform and no modified residues or glycosylation sites, so it does not identify a cause for extra bands. Confirm their identity before assigning them to LIG4.
LIG4 is listed as nuclear. Check the nuclear fraction when comparing its signal across fractionated samples.
LIG4 is associated with DNA damage and repair, but the supplied features do not establish that damage treatment increases its abundance. Measure treated and control samples before inferring induction.
Compare them with the approximately 104-kDa expected band. The supplied record lists one isoform and no modified residues or glycosylation sites, so it does not identify a cause for extra bands. Confirm their identity before assigning them to LIG4.
Boster reagents
LIG4 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-DNA Ligase IV/LIG4 Antibody Picoband®
Cat # A01685-3
Real WB data
Anti-DNA Ligase IV Rabbit Monoclonal Antibody
Cat # M01685
Two the supplier anti-LIG4 antibodies have WB images. A01685-3 shows an approximately 104 kDa band in the listed human cell and rat and mouse testis lysates under reported conditions; M01685 has a Ramos lysate WB image with limited methodological detail.
Which to pick: Choose A01685-3 for the documented human, mouse, or rat sample contexts and a detailed WB protocol. M01685 is listed for human reactivity and has a Ramos lysate WB image; its supplied caption gives fewer experimental details.
Source: BosterBio LIG4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P49917.
- Human Protein Atlas. LIG4 tissue expression.
- PMC6305145 — target-verified WB comparison
- PMC12184210 — target-verified WB comparison
- PMC6134031 — target-verified WB comparison