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EIF4E / Eukaryotic translation initiation factor 4E · Western blot design guide
Design a Western Blot for EIF4E
Real validated EIF4E Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EIF4E 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
EIF4E 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 | ~25.1 kDa | |
| Observed band | ~25 kDa | |
| Gel | 12% (catalog P00135) | |
| Positive control | Cerebellum (total-target IHC; phospho state unverified) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Activation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated EIF4E Western Blot Protocols
The P00135 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human HepG2, human MCF-7 (catalog P00135) |
| Gel % | 12% (catalog P00135) |
| Load | 30 ug; reducing conditions (catalog P00135) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog P00135) |
| Membrane | nitrocellulose membrane (catalog P00135) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog P00135) |
| Primary antibody | P00135 · 1:1000 (catalog P00135) |
| Primary incubation | overnight at 4°C (catalog P00135) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog P00135) |
| Secondary incubation | 1.5 hour at RT (catalog P00135) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog P00135) |
| Detection | ECL (catalog P00135) |
Section 2
What Is the Expected EIF4E Western Blot Band Size?
EIF4E is predicted at 25.1 kDa and observed near 25 kDa; isoform-specific migration is unestablished, so confirm band identity and phospho-S209 specificity.
| Band near 25 kDa | Consistent with eIF4E; antibody specificity should confirm identity |
| Band near 25 kDa with a phospho-S209 antibody | Consistent with eIF4E phosphorylated at Ser209 |
| No band near 25 kDa with a phospho-S209 antibody | Ser209 phosphorylation may be absent or below detection |
| Several bands at different positions | Isoforms 1, 2, and 3 are documented, but their migration and band identities are unverified |
💡Expected EIF4E appearanceUniProt predicts 25.1 kDa, and antibody QC shows a band at approximately 25 kDa; confirm its identity with antibody specificity controls, especially for the phospho-S209 antibody.
| UniProt predicted mass | Predicts 25.1 kDa; the observed band is approximately 25 kDa |
| Isoform 1 | Its apparent size relative to the other isoforms is not supplied |
| Isoform 2 | Its apparent size relative to the other isoforms is not supplied |
| Isoform 3 | Its apparent size relative to the other isoforms is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | The phospho-S209 antibody may have no detectable phosphorylated target | Check total eIF4E and a phospho-S209 positive control |
| Band higher than expected | Its identity or relationship to the documented isoforms is unverified | Compare with total eIF4E and confirm specificity by EIF4E depletion |
| Band lower than expected | Its identity or relationship to the documented isoforms is unverified | Confirm specificity by EIF4E depletion and compare with total eIF4E |
| Multiple bands | EIF4E has three named isoforms, but distinct band positions are not established | Confirm which bands depend on EIF4E and assess isoform identity |
| Weak or no signal | Ser209 phosphorylation may be low when using the phospho-specific antibody | Check total eIF4E and a phospho-S209 positive control |
Experiment evidence
Western blot images showing EIF4E band
P00135 · EIF4E
P00135 · EIF4E
M00135 · EIF4E
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EIF4E Western blot
🧪HPA-IHC candidate guidance (verify in WB): HPA tissue expression does not establish phosphorylation at the selected antibody epitope. Verify activation-state controls for the phospho-specific lead. HPA-IHC candidate guidance (verify in WB): For positive controls for EIF4E in Western blot, you can use cerebellum lysate, which has high HPA expression.
Positive control: Cerebellum (total-target IHC; phospho state unverified)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No HPA tissue is listed as not detected, so use siRNA knockdown or a KO line for a clear negative control.
HPA tissue expression evidence for EIF4E
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 |
|---|---|---|---|---|
| Cerebellum | GLUC cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Lung | alveolar cells type I | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced EIF4E Western Blot Tips
Deeper troubleshooting and optimisation questions for EIF4E, answered from its protein features.
Where should the main EIF4E band appear?
Band shift · Canonical EIF4E has a predicted mass of 25.1 kDa, consistent with the supplied observed band near 25 kDa. Use that region to identify a candidate band, then confirm its identity with an EIF4E-specific antibody.
Could EIF4E isoforms produce additional bands?
Isoforms · UniProt lists three isoforms. Isoform 3 replaces canonical residues 1–6 with a longer N-terminal sequence; isoform 2 replaces canonical residue 133 with a longer sequence. Either change could affect apparent size or antibody recognition, but these features alone do not establish that separate bands will be visible.
Yes, depending on its binding site. Isoform 3 changes the canonical N-terminal residues 1–6, while isoform 2 changes the sequence beginning at canonical residue 133. Check the antibody epitope against all three sequences before interpreting a missing or weaker isoform band.
Yes, depending on its binding site. Isoform 3 changes the canonical N-terminal residues 1–6, while isoform 2 changes the sequence beginning at canonical residue 133. Check the antibody epitope against all three sequences before interpreting a missing or weaker isoform band.
Which EIF4E modifications matter when choosing a phospho-specific antibody?
PTM · The supplied UniProt coordinates list phosphothreonine at position 22 and phosphoserine at position 209; PKC and MKNK2 are listed for position 209. Check the antibody’s stated site and numbering convention against these UniProt coordinates, especially if it uses numbering from a paper or a different isoform.
Phosphorylation is listed at UniProt positions 22 and 209, and N-acetylalanine at position 2. Their presence does not establish a visible mobility shift or explain a difference between observed and calculated mass. Compare any shifted signal with a total EIF4E blot before assigning it to a modification.
Measure the site-specific phospho signal alongside total EIF4E from comparable samples. Report which site the antibody recognizes and keep its numbering convention explicit. A change in phospho signal alone cannot distinguish altered phosphorylation from a change in total EIF4E abundance.
Phosphorylation is listed at UniProt positions 22 and 209, and N-acetylalanine at position 2. Their presence does not establish a visible mobility shift or explain a difference between observed and calculated mass. Compare any shifted signal with a total EIF4E blot before assigning it to a modification.
Measure the site-specific phospho signal alongside total EIF4E from comparable samples. Report which site the antibody recognizes and keep its numbering convention explicit. A change in phospho signal alone cannot distinguish altered phosphorylation from a change in total EIF4E abundance.
Does this guide establish induction of EIF4E?
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 EIF4E?
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 P00135 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 EIF4E be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
How should unexpected EIF4E bands be interpreted?
Interpretation · First compare them with the expected region near 25 kDa. The two listed alternative sequences make isoform recognition a possibility; the listed modifications do not, by themselves, identify another band. Check which EIF4E sequence the antibody recognizes before labeling an unexpected band as an isoform or modified EIF4E.
Boster reagents
EIF4E 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-Phospho-eIF4E (S209) Rabbit Monoclonal Antibody
Cat # P00135
Real WB data
Anti-EIF4E Monoclonal Antibody
Cat # M00135
The catalog reports two EIF4E Western blot antibodies: one against EIF4E and one against phospho-eIF4E (S209). Each has a WB image showing an approximately 25 kDa band in named human, rat, and mouse lysates. The supplied evidence does not establish performance beyond those tested contexts.
Which to pick: Choose M00135 for EIF4E detection or P00135 for the S209 phosphorylated form. Both have WB images from human, rat, and mouse samples; the reported primary dilutions are 1:100 and 1:1000, respectively.
Source: BosterBio EIF4E gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P06730.
- Human Protein Atlas. EIF4E tissue expression.
- PMC549047 — target-verified WB comparison
- PMC4558139 — target-verified WB comparison
- PMC10866396 — target-verified WB comparison