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- Table of Contents
EIF3D / Eukaryotic translation initiation factor 3 subunit D · Western blot design guide
Design a Western Blot for EIF3D
Source-linked EIF3D Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EIF3D 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
EIF3D 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 | ~64 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Tonsil (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 + Acetylated | |
| Caveat | Peptide-blocking control | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked EIF3D Western Blot Protocol Options
The A30683 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysates from NIH/3T3 cells, (catalog A30683) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | the synthesized peptide (catalog A30683) |
| Primary antibody | A30683; use the WB datasheet starting dilution (standard starting point) |
| 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 EIF3D Western Blot Band Size?
EIF3D is predicted at 64 kDa; its isoforms may affect migration, but no empirical band size or distinct isoform pattern is established.
| Single band near 64 kDa | consistent with predicted EIF3D mass |
| One band despite three isoforms | isoforms may overlap in migration or detection |
| Several discrete bands | isoforms 1, 2, and 3 are possible contributors; band identities need confirmation |
| Closely spaced doublet | possible isoform migration differences, without an established pattern |
💡Expected EIF3D appearanceEIF3D has a predicted mass of about 64 kDa, but no empirical band size or isoform migration pattern is supplied; confirm any candidate band with peptide blocking or another identity control.
| UniProt predicted mass | places the reference size near 64 kDa, without establishing apparent migration |
| Splice isoform 1 | its individual mass and migration are not supplied |
| Splice isoform 2 | may migrate differently from other isoforms, but its size is not supplied |
| Splice isoform 3 | may migrate differently from other isoforms, but its size is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EIF3D signal may be below detection in this lysate | check loading and enrichment of the cytoplasmic fraction |
| Band higher than expected | apparent migration or band identity is uncertain | compare with a molecular weight marker and a peptide-blocked lane |
| Band lower than expected | an isoform is possible, but its size is unknown | confirm identity by peptide blocking or EIF3D depletion |
| Multiple bands | isoforms 1, 2, and 3 are possible contributors | confirm each candidate band with an identity control |
| Weak or no signal | cytoplasmic EIF3D may be below assay detection | check sample loading and antibody performance with a positive lysate |
Experiment evidence
Western blot images showing EIF3D band
A30683 · EIF3D
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EIF3D Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EIF3D in Western blot, you can use tonsil tissue, which HPA rates as highly positive.
Positive control: Tonsil (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: EIF3D is cytoplasmic, and HPA reports adipose tissue as not detected, making tissue controls feasible.
HPA tissue expression evidence for EIF3D
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 |
|---|---|---|---|---|
| Tonsil | non-germinal center cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular 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 → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced EIF3D Western Blot Tips
Deeper troubleshooting and optimisation questions for EIF3D, answered from its protein features.
Where should the main EIF3D band appear?
Band shift · The predicted mass is 64 kDa. No observed band position is supplied, so use 64 kDa as a starting point, not a guaranteed apparent mass. The listed modifications alone do not establish a visible shift.
Could EIF3D isoforms produce additional bands?
Isoforms · EIF3D has three listed isoforms. Relative to isoform 1, isoform 2 lacks residues 54–102 and isoform 3 lacks residues 137–151. These differences could affect migration, but the features do not establish which isoforms are present in your sample or whether they resolve into separate bands.
For detection across all three isoforms, choose an antibody binding region outside residues 54–102 and 137–151 of the canonical sequence. An antibody targeting either deleted segment may miss the corresponding isoform. Check the antibody’s stated binding region against those coordinates.
For detection across all three isoforms, choose an antibody binding region outside residues 54–102 and 137–151 of the canonical sequence. An antibody targeting either deleted segment may miss the corresponding isoform. Check the antibody’s stated binding region against those coordinates.
Could phosphorylation affect EIF3D band interpretation?
PTM · UniProt lists phosphoserine at canonical residues 161, 528 and 529. If a band pattern suggests phosphorylation, compare matched samples with and without phosphatase treatment. These sites do not, by themselves, demonstrate a visible band shift; check the numbering convention before comparing them with antibody or paper coordinates.
Yes. UniProt lists N6-acetyllysine at canonical residue 53. Use that coordinate when checking a modification-specific antibody or comparing site reports. The listed site alone does not establish its occupancy in your sample or explain a band shift.
Yes. UniProt lists N6-acetyllysine at canonical residue 53. Use that coordinate when checking a modification-specific antibody or comparing site reports. The listed site alone does not establish its occupancy in your sample or explain a band shift.
Does this guide establish induction of EIF3D?
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 EIF3D?
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 A30683 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 EIF3D bands be quantified across samples?
Quantitation · Use an antibody binding region shared by the isoforms you intend to measure, and apply the same band-inclusion rule across samples. Isoforms 2 and 3 have different deletions, so quantifying one band may not capture all EIF3D forms. The supplied features do not identify which isoforms are expressed in a given sample.
How should unexpected EIF3D bands be assessed?
Interpretation · Compare their positions with the 64 kDa predicted mass and consider the listed isoform deletions. Check whether the antibody binding region is present in each isoform. Phosphorylation at canonical residues 161, 528 and 529 and acetylation at 53 are documented, but their presence alone cannot assign an unexpected band to a modification.
Boster reagents
EIF3D 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-EIF3D Antibody
Cat # A30683
The catalog reports one anti-EIF3D antibody for Western blot, A30683, with stated human and mouse reactivity. Its WB image shows NIH/3T3 cell lysate and a lane blocked with synthesized peptide; the supplied evidence does not show a human sample.
Which to pick: A30683 is the only listed option. Its WB image documents testing in NIH/3T3 cells; human reactivity is listed, but no human WB example is supplied.
Source: BosterBio EIF3D gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15371.
- Human Protein Atlas. EIF3D tissue expression.
- PMC9937894 — target-verified WB comparison
- PMC6945244 — target-verified WB comparison
- PMC12678802 — target-verified WB comparison