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- Table of Contents
CD3D / T-cell surface glycoprotein CD3 delta chain · Western blot design guide
Design a Western Blot for CD3D
Real validated CD3D Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CD3D 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
CD3D 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 | ~18.9 kDa | |
| Observed band | ~19 kDa | |
| Gel | 5–20% (catalog A04405-2) | |
| Positive control | Kidney (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CD3D Western Blot Protocols
The A04405-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human CCRF-CEM, human MOLT-4 (catalog A04405-2) |
| Gel % | 5–20% (catalog A04405-2) |
| Load | 30 ug; reducing conditions (catalog A04405-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04405-2) |
| Membrane | nitrocellulose membrane (catalog A04405-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04405-2) |
| Primary antibody | A04405-2 · 0.5 μg/mL (catalog A04405-2) |
| Primary incubation | overnight at 4°C (catalog A04405-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04405-2) |
| Secondary incubation | 1.5 hour at RT (catalog A04405-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04405-2) |
| Detection | ECL (catalog A04405-2) |
Section 2
What Is the Expected CD3D Western Blot Band Size?
CD3D is predicted at 18.9 kDa and observed at ~19 kDa; the cause of any difference is not established.
| Band at ~19 kDa | Empirical CD3D band in reducing whole-cell lysates, close to the 18.9 kDa predicted mass |
| Band above ~19 kDa | Possible contribution from N-linked glycosylation at Asn38 or Asn74; band identity needs confirmation |
| Band below ~19 kDa | Possible signal-peptide cleavage or a smaller CD3D species; band identity needs confirmation |
| Multiple bands near the expected region | Possible CD3D isoforms 1 and 2 or glycosylated forms; distinct migration is unproven |
💡Expected CD3D appearanceCD3D has a predicted mass of 18.9 kDa and an empirical band at ~19 kDa in reducing whole-cell lysates; confirm unexpected bands with a positive control and an independent identity check.
| UniProt predicted mass | 18.9 kDa precursor prediction; the observed CD3D band is ~19 kDa |
| N-linked glycosylation at Asn38 | Could alter apparent mass if occupied; no visible shift is established |
| N-linked glycosylation at Asn74 | Could alter apparent mass if occupied; no visible shift is established |
| Signal peptide at residues 1–21 | Cleavage reduces mature-chain mass relative to the precursor; migration effect is unmeasured |
| Isoforms 1 and 2 | May differ in mass, but their relative sizes and band separation are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CD3D is a membrane protein that may be poorly extracted | Check membrane-protein extraction and run a Jurkat lysate positive control |
| Band higher than expected | Possible N-linked glycosylation at Asn38 or Asn74 | Compare treated and untreated samples after N-glycan removal and verify CD3D identity |
| Band lower than expected | Possible signal-peptide cleavage or sample proteolysis | Check sample integrity and confirm the band with an independent CD3D antibody |
| Broad smear instead of sharp band | Possible heterogeneous glycosylation at the two N-linked sites | Compare N-glycan-treated and untreated samples and verify CD3D identity |
| Multiple bands | Possible isoforms 1 and 2 or different glycosylation states | Compare N-glycan-treated samples and confirm bands with an independent CD3D antibody |
| Weak or no signal | Poor membrane-protein recovery or low CD3D in the sample | Check extraction and compare with Jurkat, CCRF-CEM, or MOLT-4 lysate |
Experiment evidence
Western blot images showing CD3D band
A04405-2 · CD3D
M04405 · CD3D
A04405-1 · CD3D
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CD3D Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CD3D in Western blot, you can use kidney tissue, the top HPA positive candidate.
Positive control: Kidney (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: As a membrane protein, CD3D may require efficient membrane extraction for a clear signal.
HPA tissue expression evidence for CD3D
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 |
|---|---|---|---|---|
| Kidney | distal tubules | Medium | Protein (IHC) | HPA → |
| Thymus | cortical cells | Medium | Protein (IHC) | HPA → |
| Tonsil | non-germinal center cells | Medium | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD3D Western Blot Tips
Deeper troubleshooting and optimisation questions for CD3D, answered from its protein features.
How should CD3D 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 CD3D isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, canonical residues 92–136 are replaced by T. Check whether the antibody epitope lies in that region before interpreting a missing or additional band. The sequence difference does not establish where either isoform will migrate.
How should glycosylation affect band interpretation?
PTM · CD3D has N-linked glycosylation sites at UniProt positions 38 and 74. If testing glycosylation as a cause of band variation, compare matched treated and untreated samples. The listed sites alone do not predict the size or visibility of a band shift.
UniProt lists phosphotyrosine at canonical positions 149 and 160. Match a phospho-specific antibody to its stated site and numbering convention; antibody or paper numbering may differ from UniProt coordinates. Compare phospho signal with total CD3D when assessing changes.
UniProt lists phosphotyrosine at canonical positions 149 and 160. Match a phospho-specific antibody to its stated site and numbering convention; antibody or paper numbering may differ from UniProt coordinates. Compare phospho signal with total CD3D when assessing changes.
Does this guide establish induction of CD3D?
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 CD3D Western blot?
Transfer · CD3D is a single-pass membrane protein with a predicted mass of 18.9 kDa. Choose transfer conditions that retain proteins near 19 kDa on the membrane, and check transfer recovery before interpreting a weak signal. The supplied features do not specify a transfer method or settings.
How should blocking be optimized for CD3D?
Blocking · CD3D has listed phosphotyrosine sites at UniProt positions 149 and 160. For a phospho-specific blot, compare blockers using the same samples and antibody, then choose the one with the clearest specific signal and lowest background. The supplied features do not identify a preferred blocker.
How can CD3D bands be quantified consistently?
Quantitation · Use the same antibody epitope and sample preparation across comparisons, since CD3D has two isoforms and two listed glycosylation sites. For phospho-CD3D measurements, assess the phospho signal alongside total CD3D; UniProt lists phosphotyrosine at canonical positions 149 and 160.
Why might CD3D migrate near 19 kDa?
Interpretation · The observed band near 19 kDa is close to the predicted 18.9 kDa. CD3D also has a signal peptide at UniProt positions 1–21 and N-linked glycosylation sites at positions 38 and 74. These features can affect the processed protein, but their presence alone does not establish a visible shift or explain an observed-versus-calculated mass difference.
Check antibody recognition of isoforms 1 and 2, whose sequences differ at canonical positions 92–136. Also consider the signal peptide at positions 1–21, N-linked sites at 38 and 74, and the listed disulfide bond when comparing sample preparations. None of these features alone identifies an unexpected band.
Check antibody recognition of isoforms 1 and 2, whose sequences differ at canonical positions 92–136. Also consider the signal peptide at positions 1–21, N-linked sites at 38 and 74, and the listed disulfide bond when comparing sample preparations. None of these features alone identifies an unexpected band.
Boster reagents
CD3D 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-CD3D Antibody Picoband®
Cat # A04405-2
Real WB data
Anti-CD3D Rabbit Monoclonal Antibody
Cat # M04405
Real WB data
Anti-CD3D Antibody
Cat # A04405-1
Three the supplier anti-CD3D antibodies list human reactivity and show Western blot images. Their captions document human cell lysates; no publication evidence is supplied. These examples establish tested contexts, not broad sample validation.
Which to pick: For a documented 19 kDa band across Jurkat, CCRF-CEM and MOLT-4 lysates, consider A04405-2. M04405 shows approximately 21 kDa versus 19 kDa expected; A04405-1 shows a Jurkat blot without a reported band size.
Source: BosterBio CD3D gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P04234.
- Human Protein Atlas. CD3D tissue expression.
- PMC12311959 — target-verified WB comparison
- PMC12528613 — target-verified WB comparison