REST / RE1-silencing transcription factor · Western blot design guide

Design a Western Blot for REST

Real validated REST Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-REST 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 REST: expected band ~121.9 kDa, hero antibody M03852, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **REST** Western blot protocol sheet — expected band ~121.9 kDa, antibody M03852, controls and PMC citations. Open the full REST WB guide →

Navigate to: Protocols Band Controls Antibodies

REST 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	~121.9 kDa
	Gel	8–10% (standard starting point)
	Positive control ⓘ	Bronchus (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	Alternative isoforms
	Gene-set association	MSigDB C7 membership
	Isoform	4 isoform(s)

Section 1

Real Curated REST Western Blot Protocols

The M03852 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	HeLa cell lysate (catalog M03852)
Gel %	8–10% (standard starting point)
Load	20–30 µg total protein per lane; optimize for abundance (standard starting point)
Transfer	Wet/tank transfer; optimize duration (standard starting point)
Membrane	0.45 µm PVDF (standard starting point)
Blocking	5% milk or 5% BSA in TBST (standard starting point)
Primary antibody	M03852; 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 REST Western Blot Band Size?

UniProt predicts a mass of 121.9 kDa for full-length REST. This is a sequence-based value, not a measured Western blot band position. The selected antibody caption identifies a HeLa lysate but reports no observed molecular mass.

What am I looking at on my blot?
Signal near the predicted full-length mass	Could be consistent with full-length REST; confirm identity with antibody specificity and appropriate controls.
Lower-mass signal	Could reflect an alternative isoform or another source of signal; size alone cannot identify it.
Several bands	Evaluate each separately using antibody epitope information and suitable controls. UniProt lists four REST isoforms.
Higher-mass signal	Assess its identity experimentally. UniProt lists REST modifications, but does not establish the cause of a higher band.

💡Expected REST appearanceUniProt predicts 121.9 kDa for full-length REST and lists four isoforms. The selected antibody caption reports no observed molecular mass, so assign bands only after checking antibody specificity and appropriate controls.

How each factor affects band size
Predicted REST mass	UniProt gives 121.9 kDa for the full-length sequence; empirical gel migration may differ.
Isoform 1	The full-length reference for comparison with the 121.9 kDa predicted mass.
Isoform 2	An alternative sequence lacking residues 314–1097; whether this antibody detects it depends on the epitope.
Isoform 3	An alternative sequence lacking residues 330–1097; whether this antibody detects it depends on the epitope.
Isoform 4	An alternative sequence lacking residues 304–326; whether this antibody detects it depends on the epitope.

Why is my band missing or off?
Situation	Likely cause	Next action
Band lower than expected	REST has shorter alternative isoforms, but band identity cannot be assigned by position alone.	Check the antibody epitope against each isoform and compare with suitable isoform controls.
Multiple bands	Alternative splicing and modifications are possible contributors; other signals also require evaluation.	Assess each band with specificity and isoform controls.
Band higher than expected	UniProt annotates phosphorylation and ubiquitin-like conjugation, but neither establishes the cause of a particular shift.	Verify band identity and assess modification only with appropriate controls.
Weak or no signal	REST is reported in both nucleus and cytoplasm; abundance and recovery can vary between fractions.	Check sample preparation and compare fractions using fraction controls.
Fragments below expected size	A lower band could represent a shorter isoform or another species; its identity is unestablished.	Check whether the antibody epitope is present and compare with isoform-specific controls.

Source: UniProt Q13127 Human Protein Atlas product datasheet (M03852)

Experiment evidence

Western blot images showing REST band

M03852 · REST

A03852-3 · REST

← swipe / scroll for more images →

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

Sample controls for REST Western blot

🧪HPA-IHC candidate guidance (verify in WB): For positive controls for REST in Western blot, you can use bronchus tissue, which shows high HPA expression.

Positive control: Bronchus (IHC candidate; verify WB)

Negative control: Suggested KO / knockdown lysate

Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.

⚠️Feasibility: No tissue is listed as not detected, so use REST knockdown or a KO line for a clear negative control.

HPA tissue expression evidence for REST

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
Bronchus	respiratory epithelial cells	High	Protein (IHC)	HPA →
Cervix	squamous epithelial cells	High	Protein (IHC)	HPA →
Endometrium	glandular cells	High	Protein (IHC)	HPA →
Esophagus	squamous epithelial cells	High	Protein (IHC)	HPA →
Fallopian tube	glandular cells	High	Protein (IHC)	HPA →

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
Caudate	glial cells	Low	Protein (IHC)	HPA →
Cerebellum	cells in granular layer	Low	Protein (IHC)	HPA →
Cerebral cortex	glial cells	Low	Protein (IHC)	HPA →
Hippocampus	glial cells	Low	Protein (IHC)	HPA →
Liver	cholangiocytes	Low	Protein (IHC)	HPA →

Section 3

Advanced REST Western Blot Tips

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

How should REST 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 should REST isoforms affect band assignment?

Isoforms · UniProt lists four isoforms, including shorter alternative sequences. Compare the antibody epitope with each isoform and use suitable controls. Do not assign an isoform from band position alone.

Could REST modifications explain a shifted band?

PTM · UniProt annotates phosphoserine at residues 864 and 971 and lists ubiquitin-like conjugation. Modifications could affect migration, but these features do not establish the cause of any particular band.

Does this guide establish induction of REST?

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 should be used for REST Western blot?

Transfer · Plan transfer for the predicted full-length mass of 121.9 kDa and verify transfer with an appropriate molecular weight marker. The supplied information does not specify a membrane or transfer setting.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the M03852 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 can REST bands be quantified consistently?

Quantitation · Define and quantify the same validated band across samples, using consistent exposure and normalization. Keep distinct bands separate unless evidence supports treating them as the same species.

What molecular mass is predicted for full-length REST?

Interpretation · UniProt predicts 121.9 kDa from the full-length sequence. That value is distinct from an observed Western blot band position; the selected antibody caption gives no observed molecular mass.

Consider isoforms, modifications and antibody specificity, then test assignments with appropriate controls. REST is reported in both nucleus and cytoplasm; use fraction controls when comparing those samples.

Boster reagents

REST 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 REST / NRSF expression in HeLa cell lysate.

Anti-REST / NRSF Rabbit Monoclonal Antibody

Cat # M03852

View product →

Real WB data

Western blot analysis of REST using anti-REST antibody (A03852-3). Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human K562 whole cell 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-REST antigen affinity purified polyclonal antibody (A03852-3) 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 ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for REST at approximately 51 kDa. The expected band size for REST is at 122 kDa.

Anti-REST Antibody Picoband®

Cat # A03852-3

View product →

Two the supplier anti-REST antibodies are listed with Western blot images. M03852 shows REST/NRSF analysis in HeLa cell lysate; A03852-3 shows REST analysis with reported gel conditions. The supplied evidence does not establish performance across other samples or conditions.

Which to pick: Both listed antibodies have Western blot images. Pick M03852 if a HeLa lysate example is most relevant; consider A03852-3 if its reported 8% SDS-PAGE conditions match your setup. Reactivity beyond the described examples is not specified.

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

References

UniProt Consortium. UniProt entry Q13127.
Human Protein Atlas. REST tissue expression.
PMC9847160 — target-verified WB comparison
PMC5376953 — target-verified WB comparison
PMC13304005 — target-verified WB comparison