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- Table of Contents
EPS8 / Epidermal growth factor receptor kinase substrate 8 · Western blot design guide
Design a Western Blot for EPS8
Real validated EPS8 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EPS8 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
EPS8 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 | ~91.9 kDa | |
| Observed band | ~97 kDa | |
| Gel | 10% (catalog A02730-1) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated EPS8 Western Blot Protocols
The A02730-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human RT4, human A431, human HepG2 (catalog A02730-1) |
| Gel % | 10% (catalog A02730-1) |
| Load | 30 ug; reducing conditions (catalog A02730-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02730-1) |
| Membrane | nitrocellulose membrane (catalog A02730-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02730-1) |
| Primary antibody | A02730-1 · 0.5 μg/mL (catalog A02730-1) |
| Primary incubation | overnight at 4°C (catalog A02730-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02730-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02730-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02730-1) |
| Detection | ECL (catalog A02730-1) |
Section 2
What Is the Expected EPS8 Western Blot Band Size?
EPS8 is predicted at 91.9 kDa and observed at approximately 97 kDa on reducing blots; the cause of that difference is not established.
| Band at approximately 97 kDa | Empirical EPS8 band in reducing whole-cell lysate blots; confirm identity with antibody controls |
| Band near 91.9 kDa | Near the predicted mass of EPS8; identity still requires confirmation |
| Higher band near twice the monomer mass | Could reflect retained EPS8 homodimers if sample denaturation is incomplete |
| Additional bands near the main band | Could reflect isoforms 1 and 2, but their migration difference is unknown |
💡Expected EPS8 appearanceEPS8 has a predicted mass of 91.9 kDa and an empirical band at approximately 97 kDa in reducing whole-cell lysates; the cause of the difference is unestablished, so confirm band identity with antibody controls.
| Predicted EPS8 mass | 91.9 kDa for the reference protein sequence |
| EPS8 homodimer | Could produce a higher band if dimers persist during sample preparation; a size shift is not demonstrated |
| Isoform 1 | Its size relative to isoform 2 is not supplied |
| Isoform 2 | Its size relative to isoform 1 is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EPS8 may be depleted from a preparation that excludes the cytoplasm or cell cortex | Check sample fractionation and include a positive whole-cell lysate |
| Band higher than expected | EPS8 homodimers may persist with incomplete denaturation; the 97 kDa band also migrates above the predicted mass for an unknown reason | Compare reducing, fully denatured samples and confirm the band with an independent antibody or EPS8 depletion |
| Band lower than expected | The supplied evidence does not identify a smaller EPS8 product | Check protein integrity and confirm the band by EPS8 depletion |
| Multiple bands | Isoforms 1 and 2 exist, but distinct migration is unverified | Confirm each band with isoform-aware reagents or EPS8 depletion |
| Weak or no signal | EPS8 abundance or recovery may be low in the tested sample | Check loading and transfer, then test a positive whole-cell lysate |
Experiment evidence
Western blot images showing EPS8 band
A02730-1 · EPS8
A02730-1 · EPS8
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EPS8 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EPS8 in Western blot, you can use adrenal gland lysate, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: The listed low-expression tissues are not clean negatives, so use siRNA knockdown or a KO line.
HPA tissue expression evidence for EPS8
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal 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 → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Soft tissue | peripheral nerve | Low | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced EPS8 Western Blot Tips
Deeper troubleshooting and optimisation questions for EPS8, answered from its protein features.
How should EPS8 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.
How could EPS8 isoforms affect band interpretation?
Isoforms · Isoform 2 lacks canonical residues 1–260. An antibody targeting that region could miss isoform 2, and the two isoforms may migrate differently. Check which region the antibody recognizes before assigning bands.
Which EPS8 phosphorylation sites matter when assessing band shifts?
PTM · UniProt lists phosphoserine at 58, 476, 625, 659, 662, 685, 811 and 815; phosphothreonine at 223 and 317; and MAPK-dependent phosphothreonine at 629. These are canonical UniProt coordinates. Their presence alone does not establish a visible shift or explain the 97 kDa band.
EPS8 interacts with EGFR, which mediates EPS8 phosphorylation according to the supplied by-similarity annotation. UniProt also assigns threonine 629 phosphorylation to MAPK. If comparing signaling conditions, assess phosphorylation alongside total EPS8; these features do not guarantee a detectable band change.
EPS8 interacts with EGFR, which mediates EPS8 phosphorylation according to the supplied by-similarity annotation. UniProt also assigns threonine 629 phosphorylation to MAPK. If comparing signaling conditions, assess phosphorylation alongside total EPS8; these features do not guarantee a detectable band change.
Does this guide establish induction of EPS8?
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 EPS8?
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 A02730-1 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 I quantify EPS8 across samples?
Quantitation · Quantify the same verified band across samples and confirm that the antibody detects the intended isoform. Isoform 2 lacks residues 1–260, so an antibody against that region would not measure both isoforms. If phosphorylation is the endpoint, distinguish a site-specific signal from total EPS8.
Why does EPS8 appear near 97 kDa instead of 91.9 kDa?
Interpretation · The observed band is about 97 kDa, while the predicted mass is 91.9 kDa. The supplied features do not establish the cause of that difference. Use the observed position when identifying the band, and verify its identity experimentally.
Consider the two annotated isoforms and verify antibody recognition: isoform 2 lacks canonical residues 1–260. EPS8 is also annotated as a phosphoprotein and homodimer, but those features alone cannot identify an unexpected band or prove a mobility shift. Confirm band identity experimentally before assigning a cause.
Consider the two annotated isoforms and verify antibody recognition: isoform 2 lacks canonical residues 1–260. EPS8 is also annotated as a phosphoprotein and homodimer, but those features alone cannot identify an unexpected band or prove a mobility shift. Confirm band identity experimentally before assigning a cause.
Boster reagents
EPS8 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-EPS8 Antibody Picoband®
Cat # A02730-1
The catalog reports one anti-EPS8 antibody, A02730-1, with a Western blot image. The caption reports a band near 97 kDa (expected 92 kDa) in human cell lysates and mouse C6 and brain lysates. Catalogued reactivity is Human; broader mouse validation is not established.
Which to pick: A02730-1 is the only listed option and has a WB image. It is catalogued for Human reactivity. The image caption also includes mouse C6 and brain lysates under reducing conditions, but those examples do not establish general mouse reactivity.
Source: BosterBio EPS8 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q12929.
- Human Protein Atlas. EPS8 tissue expression.
- PMC4304094 — target-verified WB comparison
- PMC6898995 — target-verified WB comparison
- PMC5778704 — target-verified WB comparison