This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
RPN2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 · Western blot design guide
Design a Western Blot for RPN2
Source-linked RPN2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RPN2 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
RPN2 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 | ~69.3 kDa | |
| Observed band | ~69 kDa | |
| Gel | 5–20% (catalog A05006-1) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +4 more | |
| Negative control | Vagina (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked RPN2 Western Blot Protocol Options
The A05006-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human THP-1, human HepG2, human T-47D (catalog A05006-1) |
| Gel % | 5–20% (catalog A05006-1) |
| Load | 30 ug; reducing conditions (catalog A05006-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05006-1) |
| Membrane | nitrocellulose membrane (catalog A05006-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05006-1) |
| Primary antibody | A05006-1 · 0.5 μg/mL (catalog A05006-1) |
| Primary incubation | overnight at 4°C (catalog A05006-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05006-1) |
| Secondary incubation | 1.5 hour at RT (catalog A05006-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05006-1) |
| Detection | ECL (catalog A05006-1) |
Section 2
What Is the Expected RPN2 Western Blot Band Size?
RPN2 is predicted at 69.3 kDa and observed at ~69 kDa; the supplied evidence does not establish a cause for the small difference.
| Band at ~69 kDa | Empirical RPN2 band in reducing whole-cell lysates |
| Band above ~69 kDa | N-linked glycosylation at Asn106 could affect migration; a visible shift is unproven |
| Band below ~69 kDa | Signal-peptide cleavage could yield a smaller mature protein |
| Additional bands at different positions | Isoforms 1 and 2 could differ in size; distinct bands are unproven |
| Little or no band in lysate | ER membrane localization may make RPN2 difficult to extract |
💡Expected RPN2 appearanceRPN2 has a predicted mass of 69.3 kDa and an empirical band at ~69 kDa in reducing whole-cell lysates; confirm band identity with ordinary antibody and sample controls.
| UniProt predicted mass | 69.3 kDa prediction closely matches the empirical ~69 kDa band |
| N-linked glycosylation at Asn106 | Could affect migration, but a visible shift is not established |
| Signal peptide at residues 1–22 | Cleavage could make mature RPN2 smaller than its precursor |
| Isoforms 1 and 2 | Could differ in size; their relative masses and migration are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ER membrane RPN2 may be poorly extracted | Check membrane solubilization and a membrane-protein extraction control |
| Band higher than expected | Asn106 glycosylation could affect migration | Compare treated and untreated samples and verify band identity |
| Band lower than expected | Signal-peptide cleavage could reduce protein size | Check band identity with an independent RPN2 antibody |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct migration is unproven | Check isoform expression and confirm bands with an independent antibody |
| Weak or no signal | ER membrane RPN2 may be poorly recovered | Check extraction, loading, transfer, and antibody performance |
Experiment evidence
Western blot images showing RPN2 band
A05006-1 · RPN2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RPN2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RPN2 in Western blot, you can use cerebellum tissue, which HPA rates High.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Vagina (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As an ER membrane protein, RPN2 requires effective membrane solubilization for reliable detection.
HPA tissue expression evidence for RPN2
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 | Purkinje cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced RPN2 Western Blot Tips
Deeper troubleshooting and optimisation questions for RPN2, answered from its protein features.
How should RPN2 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 RPN2 isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Relative to the canonical sequence, isoform 2 lacks residues 70..101 and replaces Lys627 with KRIAAEQSSRLAKYRTL. These sequence changes could affect migration. Choose an antibody epitope with these differences in mind; distinct bands alone do not identify either isoform.
Which RPN2 modification matters when interpreting a band?
PTM · UniProt annotates N-linked glycosylation at Asn106 in the canonical sequence. If testing whether glycosylation affects migration, compare matched samples before and after deglycosylation. The site annotation does not predict the size or visibility of a shift. UniProt coordinates may differ from antibody or paper numbering.
Does this guide establish induction of RPN2?
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 RPN2 Western blot?
Transfer · RPN2 is an endoplasmic reticulum multi-pass membrane protein with an observed band near 69 kDa. Check transfer efficiency around that size when choosing transfer conditions. The supplied features do not establish a specific transfer method as optimal.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05006-1 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 RPN2 bands be quantified?
Quantitation · Quantify the band near the observed ~69 kDa position consistently across samples, and verify that signal remains within the assay's linear range. If comparing isoforms, account for the missing residues 70..101 and altered residue 627 in isoform 2 when selecting an antibody.
Why might RPN2 migrate differently from its predicted mass?
Interpretation · RPN2 has a predicted mass of 69.3 kDa and an observed band near 69 kDa. UniProt lists an N-linked glycosylation site at Asn106 and a signal peptide at residues 1..22, but these features alone do not establish a visible shift or explain a mass difference.
Consider the two annotated isoforms and N-linked glycosylation at canonical Asn106 as possibilities to test. Isoform 2 lacks canonical residues 70..101 and has a changed C-terminal sequence beginning at residue 627. Band position alone cannot establish the identity or cause of an unexpected band.
Consider the two annotated isoforms and N-linked glycosylation at canonical Asn106 as possibilities to test. Isoform 2 lacks canonical residues 70..101 and has a changed C-terminal sequence beginning at residue 627. Band position alone cannot establish the identity or cause of an unexpected band.
Boster reagents
RPN2 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-RPN2 Antibody Picoband®
Cat # A05006-1
the supplier A05006-1 is an anti-RPN2 antibody listed for human reactivity. Its Western blot image shows an approximately 69 kDa band, matching the expected size, in human THP-1, HepG2 and T-47D whole-cell lysates. Evidence for other sample types is not supplied.
Which to pick: A05006-1 is the only listed RPN2 antibody. It has a Western blot image from three human cell lines, making it the documented choice for those sample contexts. Check suitability separately for other samples.
Source: BosterBio RPN2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P04844.
- Human Protein Atlas. RPN2 tissue expression.
- PMC5880597 — target-verified WB comparison
- PMC6059750 — target-verified WB comparison
- PMC4413657 — target-verified WB comparison