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EEF1A2 / Elongation factor 1-alpha 2 · Western blot design guide
Design a Western Blot for EEF1A2
Source-linked EEF1A2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EEF1A2 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
EEF1A2 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 | ~50.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Caudate (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 | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked EEF1A2 Western Blot Protocol Options
The A03304 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of MCF-7 cells, (catalog A03304) |
| 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 | Blocking buffer: 3% nonfat dry milk in TBST (catalog A03304) |
| Primary antibody | A03304; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A03304) |
| 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 EEF1A2 Western Blot Band Size?
EEF1A2 has a predicted 50.5 kDa monomer; homodimerization and reported modifications are possible band considerations, but no empirical migration or feature-driven shift is established.
| Band near 50.5 kDa | consistent with the predicted EEF1A2 monomer; confirm identity with controls |
| Band near 101 kDa under nonreducing conditions | could reflect a homodimer that survives sample preparation |
| Faint band in a membrane-depleted fraction | may reflect EEF1A2 localization at the endoplasmic reticulum membrane |
| Band near 50.5 kDa despite modification states | reported methylation and phosphorylation do not establish a resolvable shift |
💡Expected EEF1A2 appearanceUniProt predicts a 50.5 kDa monomer; no empirical band size is supplied, and a band near this size requires ordinary identity controls.
| Predicted monomer mass | sets a reference near 50.5 kDa, without establishing observed migration |
| Homodimer formation | could yield a band near twice the monomer mass if the dimer survives sample preparation |
| Trimethylglycine at residue 2 | adds a small modification whose visible effect is not established |
| Alternate lysine methylation states at residue 36 | change mass slightly; distinct bands are not established |
| Phosphoserine at residue 163 | changes mass slightly; a visible shift is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EEF1A2 may be missed if endoplasmic reticulum membranes are poorly extracted | Check membrane extraction and a positive control |
| Band higher than expected | a homodimer may survive sample preparation | Compare reducing and nonreducing samples and verify band identity |
| Band lower than expected | the supplied features do not establish a smaller mature form | Check band identity with an independent antibody or EEF1A2 depletion |
| Weak or no signal | membrane extraction or detection may be inadequate | Check extraction, loading and a positive control |
| Multiple bands | the supplied modification sites do not establish distinct migrating forms | Compare bands using an independent antibody or EEF1A2 depletion |
Experiment evidence
Western blot images showing EEF1A2 band
A03304 · EEF1A2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EEF1A2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EEF1A2 in Western blot, you can use caudate tissue, which HPA rates as highly positive.
Positive control: Caudate (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: A tissue contrast is feasible, though ER membrane localization makes adequate protein extraction important.
HPA tissue expression evidence for EEF1A2
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | High | Protein (IHC) | HPA → |
| Adrenal gland | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EEF1A2 Western Blot Tips
Deeper troubleshooting and optimisation questions for EEF1A2, answered from its protein features.
What should I check if unexpected EEF1A2 bands appear?
Band shift · Check whether the main signal is consistent with the 50.5 kDa reference and validate the identity of any additional bands. EEF1A2 is annotated as a homodimer and has multiple modifications, but these features do not establish the cause or apparent size of an unexpected band. The supplied record has no observed Western blot band size.
Could an annotated isoform explain a second EEF1A2 band?
Isoforms · Only one isoform is supplied, with no alternative sequence. An additional band cannot be assigned to an EEF1A2 isoform from these features alone.
Which EEF1A2 modifications should I consider when interpreting bands?
PTM · UniProt lists trimethylglycine at position 2; methyllysine at 36, 55, 79, and 165; acetyllysine at 179 and 439; phosphoserine at 163 and 224; phosphothreonine at 239; and 5-glutamyl glycerylphosphorylethanolamine at 301 and 374. These are UniProt coordinates; antibody or paper numbering may differ. None establishes a visible band shift by itself.
UniProt annotates phosphoserine at positions 163 and 224 and phosphothreonine at 239. If testing whether phosphorylation contributes to a band pattern, compare appropriately matched samples with and without phosphatase treatment. The annotations alone do not show that phosphorylation produces a resolvable shift.
UniProt annotates phosphoserine at positions 163 and 224 and phosphothreonine at 239. If testing whether phosphorylation contributes to a band pattern, compare appropriately matched samples with and without phosphatase treatment. The annotations alone do not show that phosphorylation produces a resolvable shift.
Does this guide establish induction of EEF1A2?
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 EEF1A2?
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 A03304 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 EEF1A2 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.
Should EEF1A2 run exactly at its predicted 50.5 kDa?
Interpretation · Use 50.5 kDa as the sequence-based reference. EEF1A2 has several annotated modifications, but their presence alone does not establish a visible shift or predict an apparent band position. No observed band size is supplied.
The supplied UniProt features name EEF1AKMT4 for alternate mono-, di-, and trimethylation at lysine 36, and EEF1AKMT3 for trimethylation at lysine 165. Mono- and dimethylation are also listed at 165 without a named enzyme. These positions use UniProt numbering.
Use the same sample fraction and preparation across comparisons, then quantify the validated EEF1A2 signal within the assay's linear range. UniProt places EEF1A2 at the endoplasmic reticulum membrane, so differing fraction recovery could affect comparisons. The supplied features do not identify which band, if several appear, is suitable for quantitation.
The supplied UniProt features name EEF1AKMT4 for alternate mono-, di-, and trimethylation at lysine 36, and EEF1AKMT3 for trimethylation at lysine 165. Mono- and dimethylation are also listed at 165 without a named enzyme. These positions use UniProt numbering.
Use the same sample fraction and preparation across comparisons, then quantify the validated EEF1A2 signal within the assay's linear range. UniProt places EEF1A2 at the endoplasmic reticulum membrane, so differing fraction recovery could affect comparisons. The supplied features do not identify which band, if several appear, is suitable for quantitation.
Boster reagents
EEF1A2 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-Elongation factor 1-alpha 2 EEF1A2 Antibody
Cat # A03304
the supplier A03304 is an anti-EEF1A2 antibody listed for Western blot with reported Human, Mouse, and Rat reactivity. Its supplied WB image uses MCF-7 cell extracts; no Mouse or Rat WB example is provided.
Which to pick: A03304 is the only listed option. Its WB image uses 25 µg of MCF-7 extract per lane, with 3% nonfat milk in TBST for blocking and HRP goat anti-rabbit IgG at 1:10,000.
Source: BosterBio EEF1A2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q05639.
- Human Protein Atlas. EEF1A2 tissue expression.
- PMC8748977 — target-verified WB comparison
- PMC2606039 — target-verified WB comparison
- PMC7867884 — target-verified WB comparison