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- Table of Contents
DDIT4 / DNA damage-inducible transcript 4 protein · Western blot design guide
Design a Western Blot for DDIT4
Real validated DDIT4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DDIT4 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
DDIT4 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 | ~25.4 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 + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated DDIT4 Western Blot Protocols
The A02019 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human kidney tissue lysate (catalog A02019) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02019 · (A) 0.5 and (B) 1 μg/mL (catalog A02019) |
| 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 DDIT4 Western Blot Band Size?
DDIT4 is predicted at 25.4 kDa; phosphorylation and regulated degradation may affect the blot, but no empirical migration or visible shift is established.
| Band near 25.4 kDa | consistent with the predicted DDIT4 monomer |
| Band with slightly altered mobility | could reflect phosphorylation; a shift is not established |
| Nearby doublet | could reflect different phosphorylation states; band identity needs confirmation |
| Weak or absent band | could reflect proteasomal degradation of DDIT4 |
💡Expected DDIT4 appearanceDDIT4 has a predicted monomer mass of 25.4 kDa, but no empirical band size is supplied; confirm any candidate band with DDIT4 depletion and an independent antibody.
| Predicted monomer mass | 25.4 kDa is the sequence-based reference, not a measured migration |
| Phosphoserine 19 | may affect migration; no visible shift is established |
| Phosphothreonine 23 | may affect migration; no visible shift is established |
| Phosphothreonine 25 | may affect migration; no visible shift is established |
| Phosphoserine 121 | may affect migration; no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | DDIT4 may be depleted through proteasomal degradation | check loading and compare conditions that alter DDIT4 abundance |
| Band higher than expected | phosphorylation may alter mobility, but the cause is unproven | compare phosphatase-treated samples and confirm identity by DDIT4 depletion |
| Band lower than expected | the predicted mass does not establish this band's identity | confirm with DDIT4 depletion and an independent antibody |
| Multiple bands | different phosphorylation states are possible but unresolved | compare phosphatase-treated samples and check which bands decrease after DDIT4 depletion |
| Weak or no signal | proteasomal degradation may limit DDIT4 abundance | verify loading and compare samples with different DDIT4 expression |
Experiment evidence
Western blot images showing DDIT4 band
A02019 · DDIT4
A02019-2 · DDIT4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DDIT4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for DDIT4 in Western blot, you can use adrenal gland tissue, which HPA rates High.
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 such as Ponceau alongside the samples.
⚠️Feasibility: DDIT4 is intracellular, so use tissue lysates for these controls.
HPA tissue expression evidence for DDIT4
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 → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial 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 → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced DDIT4 Western Blot Tips
Deeper troubleshooting and optimisation questions for DDIT4, answered from its protein features.
How should DDIT4 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 a second DDIT4 band be a documented isoform?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. These features do not support assigning a second band to an annotated DDIT4 isoform; verify its identity before doing so.
Which DDIT4 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserines at positions 19 and 121 and phosphothreonines at positions 23 and 25. Keep those UniProt coordinates explicit when comparing antibody or paper numbering. The annotations establish modification sites, but do not show whether phosphorylation produces resolvable bands in your samples.
Does this guide establish induction of DDIT4?
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 DDIT4?
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 A02019 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 can protein turnover affect DDIT4 quantitation?
Quantitation · DDIT4 interacts with TXNIP, which inhibits its proteasomal degradation. When comparing band intensity across conditions, consider that a change in DDIT4 abundance could reflect altered stability. The supplied features do not establish which conditions change TXNIP or DDIT4 turnover.
How should DDIT4's predicted mass guide band identification?
Interpretation · DDIT4 has a predicted mass of 25.4 kDa. No empirical apparent band is supplied, so use that value as a reference rather than an expected gel position. The listed phosphorylation sites do not by themselves establish a visible shift or explain any difference from the predicted mass.
UniProt places DDIT4 in the cytosol and mitochondrion. If assessing fractions, examine both rather than treating a signal in only one as the complete DDIT4 pool. The supplied features do not specify the relative amount in either location.
Check candidate bands against the 25.4 kDa predicted mass and verify their identity experimentally. UniProt lists one isoform, four phosphorylation sites, and no glycosylation sites, signal peptide, or propeptide. None of those features alone identifies an unexpected band or proves a visible mass shift.
UniProt places DDIT4 in the cytosol and mitochondrion. If assessing fractions, examine both rather than treating a signal in only one as the complete DDIT4 pool. The supplied features do not specify the relative amount in either location.
Check candidate bands against the 25.4 kDa predicted mass and verify their identity experimentally. UniProt lists one isoform, four phosphorylation sites, and no glycosylation sites, signal peptide, or propeptide. None of those features alone identifies an unexpected band or proves a visible mass shift.
Boster reagents
DDIT4 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-RTP801 DDIT4 Antibody
Cat # A02019
Real WB data
Anti-REDD1/DDIT4 specific Antibody
Cat # A02019-2
Two the supplier anti-DDIT4 antibodies list human, mouse, and rat reactivity and show human Western blots. A02019 was tested on kidney tissue lysate; A02019-2 on K562 lysate, where the reported band was approximately 35 kDa versus an expected 25 kDa.
Which to pick: Choose A02019 for a human kidney lysate example (0.5 or 1 μg/mL), or A02019-2 for a K562 whole cell lysate example (1:1000). Both have WB images; the supplied images do not show mouse or rat samples.
Source: BosterBio DDIT4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NX09.
- Human Protein Atlas. DDIT4 tissue expression.
- PMC11979484 — target-verified WB comparison
- PMC6461855 — target-verified WB comparison
- PMC3907060 — target-verified WB comparison