RAG2 / V(D)J recombination-activating protein 2 · Western blot design guide

Design a Western Blot for RAG2

Real validated RAG2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAG2 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

Western blot protocol sheet for RAG2: expected band ~59.2 kDa, hero antibody A00352, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **RAG2** Western blot protocol sheet — expected band ~59.2 kDa, antibody A00352, controls and PMC citations. Open the full RAG2 WB guide →

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RAG2 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	~59.2 kDa
	Observed band	~55 kDa
	Gel	5–20% (catalog A00352)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	—
	Caveat	Observed below predicted
	Gene-set association	MSigDB C7 membership
	Isoform	1 isoform(s)

Section 1

Real Curated RAG2 Western Blot Protocols

The A00352 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human MOLT-4, mouse thymus, mouse spleen (catalog A00352)
Gel %	5–20% (catalog A00352)
Load	30 ug; reducing conditions (catalog A00352)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00352)
Membrane	nitrocellulose membrane (catalog A00352)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A00352)
Primary antibody	A00352 · 0.5 μg/mL (catalog A00352)
Primary incubation	overnight at 4°C (catalog A00352)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A00352)
Secondary incubation	1.5 hour at RT (catalog A00352)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A00352)
Detection	ECL (catalog A00352)

Section 2

What Is the Expected RAG2 Western Blot Band Size?

RAG2 is predicted at 59.2 kDa and observed near 55 kDa; the supplied features do not establish the cause of that difference.

What am I looking at on my blot?
Band near 55 kDa	Matches the empirical RAG2 band reported in reducing lysates; confirm identity with controls
Band near 59.2 kDa	Near the UniProt predicted sequence mass
Single sharp band near the expected size	Consistent with RAG2, which has no listed glycosylation sites or additional isoforms
Band enriched in a nuclear fraction	Consistent with RAG2 nuclear localization

💡Expected RAG2 appearanceUniProt predicts 59.2 kDa, while antibody QC reports a band near 55 kDa; the supplied features do not establish why they differ, so confirm identity with appropriate controls.

How each factor affects band size
UniProt predicted molecular weight	Sets a sequence-based reference of 59.2 kDa
Full-length predicted mass	Provides a reference for assessing the empirical band near 55 kDa
Sequence-based mass of 59,241 Da	Corresponds to approximately 59.2 kDa before accounting for apparent migration
Predicted size of the 527-residue protein	Does not establish the cause of the reported 55 kDa migration

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear RAG2 may be poorly recovered	Check nuclear extraction and run a known positive lysate
Band higher than expected	Band identity or apparent migration is uncertain	Compare with the reported 55 kDa band and verify specificity with a second antibody
Band lower than expected	Degradation or nonspecific binding is possible	Use fresh protease-inhibited lysate and confirm with a second antibody
Multiple bands	The supplied features do not establish additional isoforms or modifications	Compare with a known positive sample and verify band identity with a second antibody
Weak or no signal	Low nuclear target recovery or limited assay sensitivity	Check nuclear recovery, loading, and transfer
Fragments below expected size	Sample degradation is possible	Prepare fresh protease-inhibited lysate and compare with a known positive sample

Source: UniProt P55895 Human Protein Atlas product datasheet (A00352)

Experiment evidence

Western blot images showing RAG2 band

A00352 · RAG2

PB9793 · RAG2

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for RAG2 Western blot

🧪For positive controls for RAG2 in Western blot, you can use an HPA-IHC candidate positive sample once one is identified; none was supplied.

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: RAG2 is nuclear, but the supplied HPA data cannot establish suitable tissue controls.

HPA tissue expression evidence for RAG2

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 RAG2 Western Blot Tips

Deeper troubleshooting and optimisation questions for RAG2, answered from its protein features.

How should RAG2 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 another RAG2 isoform explain an unexpected band?

Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no isoform-based explanation for another band.

Do listed modifications explain a RAG2 band shift?

PTM · No modified residues or glycosylation sites are listed. The supplied features therefore do not support assigning a band shift to a specific modification.

Does this guide establish induction of RAG2?

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 RAG2?

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 A00352 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 RAG2 be quantified?

Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.

Why might RAG2 appear near 55 kDa instead of 59.2 kDa?

Interpretation · The supplied apparent band is about 55 kDa, while the predicted mass is 59.2 kDa. These values differ, but the listed features do not establish why. Confirm the band with a RAG2-specific control before assigning it.

RAG2 is listed as nuclear. Prioritize a nuclear-containing sample and keep fraction preparation consistent when comparing lanes.

RAG2 is a component of a complex with RAG1 and associated HMGB1 or HMGB2. That association alone does not identify a higher Western-blot band; verify its RAG2 specificity.

Because RAG2 is listed as nuclear, compare consistently prepared nuclear-containing samples and account for differences in nuclear sample loading before interpreting band intensity.

Boster reagents

RAG2 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

Western blot analysis of RAG2 using anti-RAG2 antibody (A00352). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human MOLT-4 whole cell lysates, Lane 2: mouse thymus tissue lysates, Lane 6: mouse spleen tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RAG2 antigen affinity purified polyclonal antibody (Catalog # A00352) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RAG2 at approximately 55 kDa. The expected band size for RAG2 is at 59 kDa.

Anti-RAG2 Antibody Picoband®

Cat # A00352

View product →

Real WB data

Western blot analysis of RAG2 using anti-RAG2 antibody (PB9793). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 40 ug of sample under reducing conditions. Lane 1: A549 Whole Cell Lysate, Lane 2: 22RV1 Whole Cell Lysate, Lane 3: U20S Whole Cell Lysate. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RAG2 antigen affinity purified polyclonal antibody (Catalog # PB9793) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for RAG2 at approximately 59 kDa. The expected band size for RAG2 is at 59 kDa.

Anti-RAG2 Antibody Picoband®

Cat # PB9793

View product →

Both listed anti-RAG2 antibodies are rabbit polyclonals with Western blot images. A00352 shows a band near 55 kDa in human MOLT-4 and mouse thymus and spleen lysates; PB9793 shows a band near 59 kDa in the listed cell lysates. No orthogonal validation is supplied.

Which to pick: Choose A00352 when a mouse tissue WB example is useful. PB9793 lists rat reactivity, but its supplied WB image uses cell lysates only; rat WB performance is not shown. Both list human and mouse reactivity.

Source: BosterBio RAG2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P55895.
Human Protein Atlas. RAG2 tissue expression.
PMC12682277 — target-verified WB comparison
PMC3304103 — target-verified WB comparison
PMC13107022 — target-verified WB comparison