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- Table of Contents
RPE65 / Retinoid isomerohydrolase · Western blot design guide
Design a Western Blot for RPE65
Real validated RPE65 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RPE65 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
RPE65 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 | ~60.9 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
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
Real Curated RPE65 Western Blot Protocols
The M00824 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | mouse eyeball lysate (catalog M00824) |
| 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 | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M00824; 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 RPE65 Western Blot Band Size?
RPE65 is predicted at 60.9 kDa; its listed modifications and membrane association may affect detection, but distinct migration effects are not demonstrated.
| Band near 60.9 kDa | Consistent with the predicted RPE65 mass; confirm identity with controls |
| Weak band in soluble lysate | RPE65 can associate with membranes when palmitoylated |
| Band in a membrane fraction | Consistent with the lipid-anchored form of RPE65 |
| Faint band in a soluble fraction | Consistent with unpalmitoylated, soluble RPE65 |
💡Expected RPE65 appearanceRPE65 has a predicted mass of 60.9 kDa, but no empirical band size is supplied; use antibody specificity and band-identity controls to assess any detected band.
| Predicted RPE65 mass | Sets a 60.9 kDa sequence-based reference, not a validated apparent band size |
| Phosphothreonine at residues 101 and 105 | Adds phosphate groups; a visible mobility change is not established |
| Phosphoserine at residue 117 | Adds a phosphate group; a distinct band is not established |
| N-acetylserine at residue 2 | Adds an acetyl group; a visible size change is not established |
| N6-acetyllysine at residue 113 | Adds an acetyl group; a visible size change is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated RPE65 may be poorly recovered | Check membrane extraction and a suitable positive lysate |
| Band higher than expected | The supplied features do not establish the cause of a large shift | Compare with a positive control and confirm antibody specificity |
| Band lower than expected | The supplied features do not establish a cleavage product | Check sample integrity and confirm band identity |
| Multiple bands | A single annotated isoform does not explain distinct bands | Compare fractions and validate bands with an independent antibody |
| Weak or no signal | Recovery may vary with membrane association | Check soluble and membrane fractions and verify the positive control |
Experiment evidence
Western blot images showing RPE65 band
M00824 · RPE65
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RPE65 Western blot
🧪For positive controls for RPE65 in Western blot, you can use no HPA-supported sample from the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA provides no tissue controls here, and lipid anchoring may affect which cell fraction contains RPE65.
HPA tissue expression evidence for RPE65
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced RPE65 Western Blot Tips
Deeper troubleshooting and optimisation questions for RPE65, answered from its protein features.
How should RPE65 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 alternative isoforms explain multiple RPE65 bands?
Isoforms · The supplied features list one isoform and no alternative sequence. Do not assign additional bands to annotated isoforms on this evidence alone.
Which RPE65 modifications matter when interpreting a blot?
PTM · In UniProt numbering, the listed sites are N-acetylserine 2, phosphothreonine 101 and 105, N6-acetyllysine 113, and phosphoserine 117. These annotations do not establish that separate bands will be visible.
Does this guide establish induction of RPE65?
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.
What transfer method to use for RPE65 Western blot?
Transfer · The supplied features do not specify a transfer method. RPE65 has a predicted mass of 60.9 kDa and may be membrane-associated or soluble. Verify transfer and recovery for the fraction being measured before comparing signals.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00824 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 RPE65 Western blot signals be quantified?
Quantitation · Keep sample fraction and light conditions consistent across comparisons. Because RPE65 can redistribute between soluble and membrane-associated forms, quantify both fractions if the goal is to assess total RPE65.
Should RPE65 run exactly at its predicted 60.9 kDa?
Interpretation · Use 60.9 kDa as a reference, not an exact band position. No observed band size is supplied. The listed modifications alone do not establish a visible shift or explain any difference from calculated mass.
RPE65 is described as membrane-associated when palmitoylated and soluble when unpalmitoylated. Check both fractions before interpreting a weak band as low total RPE65.
RPE65 interacts with MYO7A in light-dependent intracellular transport. Record light conditions and compare the same fraction across samples; a fraction-specific signal change need not indicate a change in total RPE65.
Do not label extra bands as isoforms or modification-dependent shifts without further evidence. Only one isoform is listed, no observed band size is supplied, and the annotated modifications do not establish distinct bands.
RPE65 is described as membrane-associated when palmitoylated and soluble when unpalmitoylated. Check both fractions before interpreting a weak band as low total RPE65.
RPE65 interacts with MYO7A in light-dependent intracellular transport. Record light conditions and compare the same fraction across samples; a fraction-specific signal change need not indicate a change in total RPE65.
Do not label extra bands as isoforms or modification-dependent shifts without further evidence. Only one isoform is listed, no observed band size is supplied, and the annotated modifications do not establish distinct bands.
Boster reagents
RPE65 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-RPE65 Rabbit Monoclonal Antibody
Cat # M00824
The catalog reports one anti-RPE65 antibody, M00824, for Western blotting. Its product record lists human, mouse, and rat reactivity and shows a Western blot of mouse eyeball lysate; the supplied evidence does not show human or rat WB results.
Which to pick: M00824 is the only listed option. Its WB image uses mouse eyeball lysate, making that the documented sample context. Human and rat are listed as reactive, but no WB images for those species are supplied.
Source: BosterBio RPE65 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16518.
- Human Protein Atlas. RPE65 tissue expression.
- PMC7012757 — target-verified WB comparison
- PMC2733357 — target-verified WB comparison
- PMC5015590 — target-verified WB comparison