This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
EIF2A / Eukaryotic translation initiation factor 2A · Western blot design guide
Design a Western Blot for EIF2A
Real validated EIF2A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EIF2A 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
EIF2A 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 | ~65 kDa | |
| Observed band | ~65 kDa | |
| Gel | 10% (catalog M02418) | |
| Positive control | Endometrium (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 | 4 isoform(s) |
Section 1
Real Curated EIF2A Western Blot Protocols
The M02418 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Ramos, human Jurkat, human Hela (catalog M02418) |
| Gel % | 10% (catalog M02418) |
| Load | 30 ug; reducing conditions (catalog M02418) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02418) |
| Membrane | nitrocellulose membrane (catalog M02418) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02418) |
| Primary antibody | M02418 · 1:500 (catalog M02418) |
| Primary incubation | overnight at 4°C (catalog M02418) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M02418) |
| Secondary incubation | 1.5 hour at RT (catalog M02418) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02418) |
| Detection | ECL (catalog M02418) |
Section 2
What Is the Expected EIF2A Western Blot Band Size?
EIF2A is predicted at 65 kDa and observed at approximately 65 kDa; the evidence does not establish distinct migration for its splice isoforms.
| Sharp band near 65 kDa | Matches the predicted mass and the observed EIF2A band; confirm identity with a specific control |
| Band near 65 kDa with another discrete band | EIF2A has splice isoforms, but the additional band's identity needs confirmation |
| Several bands at different positions | Splice isoforms are possible contributors; their migration differences are not established |
| Single band away from 65 kDa | Could represent an isoform, but its identity is unconfirmed |
💡Expected EIF2A appearanceEIF2A has a predicted mass of 65 kDa and an observed band at approximately 65 kDa; confirm band identity with an appropriate control.
| UniProt predicted mass | Places the reference band near 65 kDa, matching the observed band |
| Splice isoform 1 | Its individual band size is not supplied |
| Splice isoform 2 | Its size relative to the other isoforms is not supplied |
| Splice isoform 3 | Its size relative to the other isoforms is not supplied |
| Splice isoform 4 | Its size relative to the other isoforms is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EIF2A signal may be below detection in the tested sample | Check loading and detection, then test a lysate with a documented EIF2A band |
| Band higher than expected | A splice isoform is possible, but its migration is unknown | Compare with the 65 kDa band and verify identity by EIF2A depletion |
| Band lower than expected | A splice isoform is possible, but its migration is unknown | Verify identity by EIF2A depletion or an independent antibody |
| Multiple bands | EIF2A has four annotated splice isoforms, without established band positions | Test which bands respond to EIF2A depletion |
| Weak or no signal | Low EIF2A abundance or insufficient assay sensitivity | Check loading and detection using a lysate with a documented EIF2A band |
Experiment evidence
Western blot images showing EIF2A band
M02418 · EIF2A
A02418-1 · EIF2A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EIF2A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EIF2A in Western blot, you can use endometrium, which HPA scores as high expression.
Positive control: Endometrium (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: HPA lists adipose tissue as not detected, but confirm low EIF2A signal in your lysate.
HPA tissue expression evidence for EIF2A
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 |
|---|---|---|---|---|
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Lung | alveolar cells type II | High | Protein (IHC) | HPA → |
| Skin | fibroblasts | High | Protein (IHC) | HPA → |
| Testis | peritubular cells | High | Protein (IHC) | HPA → |
| Appendix | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EIF2A Western Blot Tips
Deeper troubleshooting and optimisation questions for EIF2A, answered from its protein features.
How should EIF2A 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 EIF2A isoforms produce additional bands?
Isoforms · Four isoforms are listed. Relative to isoform 1, isoform 2 lacks residues 1–213 and changes 214–219; isoform 3 lacks 34–58; isoform 4 lacks 98–158. Check whether the antibody epitope is retained before assigning an extra band to an isoform.
Which EIF2A phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphothreonine at positions 5 and 518 and phosphoserine at 503, 506, 517, and 526. These are UniProt coordinates; antibody or paper numbering may differ. Their presence does not establish that phosphorylation caused a visible band shift.
Does this guide establish induction of EIF2A?
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 EIF2A?
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 M02418 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 EIF2A bands be quantified across samples?
Quantitation · Quantify the approximately 65 kDa band consistently across samples. If additional bands appear, report them separately until their identity is established. The listed isoforms and modifications provide candidates to investigate, but do not establish which bands they produce.
Should EIF2A migrate above its predicted 65 kDa mass?
Interpretation · The reported band is approximately 65 kDa, matching the predicted mass. EIF2A has documented phosphorylation and N-terminal acetylation, but those features alone do not establish a visible shift or explain a mass discrepancy.
UniProt lists N-acetylmethionine at position 1 and N-acetylalanine at position 2 in N-terminally processed EIF2A. For an antibody targeting the extreme N terminus, check which form its epitope recognizes. Isoform 2 also lacks residues 1–213.
Check their size and the antibody epitope against the listed isoforms: isoform 2 lacks 1–213, isoform 3 lacks 34–58, and isoform 4 lacks 98–158. Phosphorylation and N-terminal processing are documented, but neither proves the identity of an unexpected band.
UniProt lists N-acetylmethionine at position 1 and N-acetylalanine at position 2 in N-terminally processed EIF2A. For an antibody targeting the extreme N terminus, check which form its epitope recognizes. Isoform 2 also lacks residues 1–213.
Check their size and the antibody epitope against the listed isoforms: isoform 2 lacks 1–213, isoform 3 lacks 34–58, and isoform 4 lacks 98–158. Phosphorylation and N-terminal processing are documented, but neither proves the identity of an unexpected band.
Boster reagents
EIF2A 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-eIF2A Rabbit Monoclonal Antibody
Cat # M02418
Real WB data
Anti-EIF2A Antibody Picoband®
Cat # A02418-1
The catalog reports two anti-EIF2A antibodies with Western blot images: M02418, a rabbit monoclonal, and A02418-1, a rabbit polyclonal. Both captions report a band near the expected 65 kDa in the tested lysates. No independent publication evidence is supplied.
Which to pick: For mouse samples, choose M02418: its blot includes mouse pancreas and heart, and it lists mouse reactivity. Both have human and rat reactivity and WB images. For human or rat work, compare your sample with the lysates shown in each caption.
Source: BosterBio EIF2A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BY44.
- Human Protein Atlas. EIF2A tissue expression.
- PMC5327398 — target-verified WB comparison