SCAI / Protein SCAI · Western blot design guide

Design a Western Blot for SCAI

Real validated SCAI Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SCAI 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 SCAI: expected band ~70.4 kDa, hero antibody A04877-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **SCAI** Western blot protocol sheet — expected band ~70.4 kDa, antibody A04877-1, controls and PMC citations. Open the full SCAI WB guide →

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SCAI 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	~70.4 kDa
	Observed band	~66 kDa
	Gel	10% (catalog A04877-1)
	Positive control ⓘ	Cerebellum (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
	Caveat	Phosphorylation-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	2 isoform(s)

Section 1

Real Curated SCAI Western Blot Protocols

The A04877-1 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 Jurkat, human K562, human U20S (catalog A04877-1)
Gel %	10% (catalog A04877-1)
Load	30 ug; reducing conditions (catalog A04877-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04877-1)
Membrane	nitrocellulose membrane (catalog A04877-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A04877-1)
Primary antibody	A04877-1 · 0.5 μg/mL (catalog A04877-1)
Primary incubation	overnight at 4°C (catalog A04877-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A04877-1)
Secondary incubation	1.5 hour at RT (catalog A04877-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A04877-1)
Detection	ECL (catalog A04877-1)

Section 2

What Is the Expected SCAI Western Blot Band Size?

SCAI is predicted at 70.4 kDa, while antibody QC reports ~66 kDa; the cause of this difference is not established.

What am I looking at on my blot?
Band at ~66 kDa	Matches the empirical SCAI band; confirm identity with antibody controls.
Band near 70.4 kDa	Near the predicted SCAI mass; identity requires confirmation.
Additional bands at different positions	Could reflect isoforms 1 and 2, whose migration differences are unknown.
Band in a nuclear, cytoplasmic, or membrane fraction	Consistent with reported SCAI locations; band position alone does not establish identity.

💡Expected SCAI appearanceSCAI has a predicted mass of 70.4 kDa, while antibody QC shows a band at ~66 kDa; the cause of the difference is unestablished, so confirm identity with ordinary antibody controls.

How each factor affects band size
Predicted SCAI mass	70.4 kDa is the sequence-based reference; the empirical band is ~66 kDa.
Isoform 1	Its mass relative to isoform 2 is not supplied.
Isoform 2	Its mass relative to isoform 1 is not supplied.
Tyr64 phosphorylation site	No apparent size shift is established from this site.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	SCAI may be below detection or poorly recovered from its cellular compartments.	Check lysate preparation and use a positive control.
Band higher than expected	Its identity or migration is uncertain; no size-raising feature is established.	Compare with a positive control and verify antibody specificity.
Band lower than expected	The reported ~66 kDa band runs below the 70.4 kDa prediction for an unknown reason.	Compare with the reported band and verify identity using a second antibody or SCAI depletion.
Multiple bands	Isoforms 1 and 2 are reported, but distinct band sizes are not established.	Check which bands respond to SCAI depletion.
Weak or no signal	SCAI abundance or recovery may vary with sample and compartment.	Check loading and fraction recovery against a positive sample.

Source: UniProt Q8N9R8 Human Protein Atlas product datasheet (A04877-1)

Experiment evidence

Western blot images showing SCAI band

A04877-1 · SCAI

← swipe / scroll for more images →

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

Sample controls for SCAI Western blot

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

Positive control: Cerebellum (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 siRNA knockdown or a KO line for a negative control.

HPA tissue expression evidence for SCAI

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	cells in granular layer	High	Protein (IHC)	HPA →
Epididymis	glandular cells	High	Protein (IHC)	HPA →
Kidney	cells in tubules	High	Protein (IHC)	HPA →
Testis	cells in seminiferous ducts	High	Protein (IHC)	HPA →
Adipose tissue	adipocytes	Medium	Protein (IHC)	HPA →

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

Tissue	Cell type	Level	Evidence	Source
Caudate	neuronal cells	Low	Protein (IHC)	HPA →
Duodenum	glandular cells	Low	Protein (IHC)	HPA →
Heart muscle	cardiomyocytes	Low	Protein (IHC)	HPA →
Liver	cholangiocytes	Low	Protein (IHC)	HPA →
Lymph node	germinal center cells	Low	Protein (IHC)	HPA →

Section 3

Advanced SCAI Western Blot Tips

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

What should be checked when unexpected SCAI bands appear?

Band shift · Check antibody recognition of both isoforms and compare bands across membrane, nuclear and cytoplasmic fractions. Isoform 2 differs at canonical position 33, and phosphotyrosine is listed at canonical position 64. Neither feature alone identifies an unexpected band or proves a mobility shift.

Could SCAI isoforms produce different bands?

Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, the arginine at canonical position 33 is replaced by SIRGSGDSSHSQSQGERYQHFTEK, making its sequence longer. Check whether the antibody recognizes both isoforms; the sequence change alone does not establish that they resolve as separate bands.

How should SCAI phosphorylation affect band interpretation?

PTM · UniProt lists phosphotyrosine at position 64 in canonical sequence numbering. Record which numbering convention an antibody uses, since isoform or antibody numbering may differ. This site supports testing for phosphorylation, but does not establish a visible shift or explain the 66 kDa band.

For an assay targeting UniProt phosphotyrosine 64, compare blockers for background and phospho-specific signal, with total SCAI measured alongside. The supplied features do not identify a preferred blocker or establish phosphorylation in a particular sample.

Does this guide establish induction of SCAI?

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 SCAI Western blot?

Transfer · SCAI is predicted at 70.4 kDa and annotated as a single-pass membrane protein that also occurs in the nucleus and cytoplasm. Verify transfer with total-protein staining and assess recovery from the fraction being tested. The supplied features do not identify one optimal transfer method.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A04877-1 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 SCAI be quantified across cellular fractions?

Quantitation · SCAI is annotated in membrane, nuclear and cytoplasmic locations. Quantify bands within each fraction using an appropriate loading measure, and assess fraction purity before comparing localization. Nuclear localization is required for its inhibition of MRTFA, so whole-cell abundance alone cannot establish that activity.

Why might SCAI appear near 66 kDa rather than 70.4 kDa?

Interpretation · The reported apparent band is about 66 kDa, while the predicted mass is 70.4 kDa. These values differ, but the supplied features do not establish why. Confirm band identity before attributing the difference to an isoform or modification.

Boster reagents

SCAI 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 SCAI using anti-SCAI antibody (A04877-1). Electrophoresis was performed on a 10% 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 Jurkat whole cell lysates, Lane 2: human K562 whole cell lysates, Lane 3: human U20S whole cell lysates, Lane 4: human U251 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: mouse brain 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-SCAI antigen affinity purified polyclonal antibody (A04877-1) 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 SCAI at approximately 66 kDa. The expected band size for SCAI is at 70 kDa.

Anti-SCAI Antibody Picoband®

Cat # A04877-1

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The catalog reports one anti-SCAI antibody for Western blotting: A04877-1, with reported human, mouse, and rat reactivity. Its WB image shows a band near 66 kDa versus an expected 70 kDa; the supplied evidence does not establish orthogonal validation.

Which to pick: A04877-1 is the only listed option and has a WB image using human Jurkat, K562, U20S, and U251 cell lysates plus rat and mouse brain lysates. Match your planned sample to those reported contexts when choosing it.

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

References

UniProt Consortium. UniProt entry Q8N9R8.
Human Protein Atlas. SCAI tissue expression.
PMC7750902 — target-verified WB comparison
PMC7848259 — target-verified WB comparison
PMC7445517 — target-verified WB comparison