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- Table of Contents
RAG1 / V(D)J recombination-activating protein 1 · Western blot design guide
Design a Western Blot for RAG1
Real validated RAG1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAG1 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
RAG1 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 | ~119.1 kDa | |
| Observed band | ~119 kDa | |
| Gel | 8% (catalog A00307-1) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated RAG1 Western Blot Protocols
The A00307-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human K562, human A431 (catalog A00307-1) |
| Gel % | 8% (catalog A00307-1) |
| Load | 30 ug; reducing conditions (catalog A00307-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00307-1) |
| Membrane | nitrocellulose membrane (catalog A00307-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00307-1) |
| Primary antibody | A00307-1 · 0.5 μg/mL (catalog A00307-1) |
| Primary incubation | overnight at 4°C (catalog A00307-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00307-1) |
| Secondary incubation | 1.5 hour at RT (catalog A00307-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00307-1) |
| Detection | ECL (catalog A00307-1) |
Section 2
What Is the Expected RAG1 Western Blot Band Size?
RAG1 is predicted at 119.1 kDa and observed near 119 kDa; its homodimer and two isoforms allow other patterns, but their migration is unconfirmed.
| Band near 119 kDa | Matches the reported RAG1 band and its predicted monomer mass |
| Band near twice the monomer size | Could reflect a RAG1 homodimer if the complex survives sample preparation |
| Additional discrete band | Could reflect isoform 1 or 2 if they migrate differently; identity needs confirmation |
| Weak whole-cell band with a stronger nuclear-fraction band | Consistent with RAG1 nuclear localization |
💡Expected RAG1 appearanceRAG1 has a predicted mass of 119.1 kDa and an empirical band near 119 kDa; confirm band identity with appropriate controls, especially for additional bands.
| Predicted RAG1 mass | Places the monomer near 119.1 kDa, consistent with the observed ~119 kDa band |
| RAG1 homodimer | Could produce a band near twice the monomer size if the complex persists during electrophoresis |
| Splice isoform 1 | May migrate differently from isoform 2; its mass is not supplied |
| Splice isoform 2 | May migrate differently from isoform 1; its mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RAG1 may be poorly recovered in the lysate | Check nuclear extraction and compare with a nuclear-enriched sample |
| Band higher than expected | A RAG1 homodimer may persist during sample preparation | Compare fully reducing and non-reducing conditions |
| Band lower than expected | An isoform could differ in size, but isoform masses are unavailable | Confirm identity with an independent antibody or RAG1 depletion |
| Multiple bands | Isoforms 1 and 2 could contribute if their migration differs | Use RAG1 depletion or isoform-specific controls to assign bands |
| Weak or no signal | Recovery of nuclear RAG1 may be low | Check nuclear enrichment and include a positive control lysate |
Experiment evidence
Western blot images showing RAG1 band
A00307-1 · RAG1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAG1 Western blot
🧪For positive controls for RAG1 in Western blot, you can use no HPA-IHC candidate positive 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 expression data here, so suitable tissue controls cannot be established.
HPA tissue expression evidence for RAG1
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 RAG1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RAG1, answered from its protein features.
How should RAG1 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 RAG1 isoforms produce different bands?
Isoforms · Two isoforms are listed. In isoform 2, canonical residues 931–1043 are replaced by one asparagine, so it has a shorter sequence. The features do not establish its apparent band position.
Check whether its epitope lies within canonical residues 931–1043. That segment is replaced in isoform 2, so an antibody directed against it may detect isoform 1 without detecting isoform 2. Confirm the antibody's stated epitope before interpreting a missing band.
Check whether its epitope lies within canonical residues 931–1043. That segment is replaced in isoform 2, so an antibody directed against it may detect isoform 1 without detecting isoform 2. Confirm the antibody's stated epitope before interpreting a missing band.
Can a modification explain an unexpected RAG1 band?
PTM · The keywords include isopeptide bond and Ubl conjugation, but the supplied features list no modified residue or site. They do not establish a visible shift or identify a modification responsible for an unexpected band.
Does this guide establish induction of RAG1?
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 RAG1?
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 A00307-1 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 RAG1 be sampled for quantitation?
Quantitation · RAG1 is listed as nuclear. Use a consistent sample preparation and compare equivalent nuclear fractions when measuring its abundance. Account for which isoform the antibody detects before comparing band intensities.
Should RAG1 migrate above its predicted mass?
Interpretation · The predicted mass is 119.1 kDa, and the supplied observed band is about 119 kDa. The listed features do not establish a shift from that value.
RAG1 is listed as a homodimer and a component of a complex with RAG2 and HMGB1 or HMGB2. Those associations suggest candidates to investigate, but the supplied features alone cannot identify an additional band or show that a complex survives the blot procedure.
RAG1 is listed as a homodimer and a component of a complex with RAG2 and HMGB1 or HMGB2. Those associations suggest candidates to investigate, but the supplied features alone cannot identify an additional band or show that a complex survives the blot procedure.
Boster reagents
RAG1 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-RAG1 Antibody Picoband®
Cat # A00307-1
A00307-1 is the listed anti-RAG1 antibody for Western blotting. Its product image reports an approximately 119 kDa band in human cell and mouse thymus and cell lysates under reducing conditions. No independent publication evidence is supplied.
Which to pick: Only A00307-1 is listed. It reports human and mouse reactivity and includes a Western blot image using Jurkat, K562, A431, mouse thymus, and RAW264.7 lysates. Compare your specimen and protocol with these reported conditions.
Source: BosterBio RAG1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P15918.
- Human Protein Atlas. RAG1 tissue expression.
- PMC8516306 — target-verified WB comparison
- PMC2705876 — target-verified WB comparison
- PMC4506145 — target-verified WB comparison