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GAS6 · Western blot design guide
Design a Western Blot for GAS6
Real validated GAS6 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GAS6 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
GAS6 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 | ~74.9 kDa | |
| Observed band | 55 kDa and 79 kDa | |
| Gel | 10–12% | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Multiple isoforms | |
| Regulation | LPS-induced | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated GAS6 Western Blot Protocols
Literature-validated Western blot parameters for GAS6 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human THP-1 , Lane 2: human A431 , Lane 3: rat brain , Lane 4: mouse brain . 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-GAS6 antigen affinity purified polyclonal antibody (A00608-1) 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 (Catalog # BA1054) 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 GAS6 at approximately 55, 79 kDa. The expected band size for GAS6 is at 79 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 55, 79 kDa |
Section 2
What Is the Expected GAS6 Western Blot Band Size?
GAS6 has a 74.9 kDa predicted backbone but typically runs as a ~79 kDa glycosylated band plus a ~55 kDa isoform-related band, reflecting N-glycosylation and alternative splicing.
| band near 79 kDa | near-full-length mature GAS6 carrying its N-linked glycan on top of the 74.9 kDa predicted backbone |
| band near 55 kDa | a shorter GAS6 species consistent with one of the five annotated splice isoforms rather than the main product |
| smeared or diffuse signal around the ~79 kDa band | heterogeneous glycan occupancy or processing at the single N420 glycosylation site |
| mature band a few kDa smaller than the full translated precursor | cleavage of the 30-residue signal peptide during secretion |
| little or no band in whole-cell lysate | GAS6 is a secreted protein, so it does not accumulate intracellularly and is mostly found in conditioned media or serum |
| more than two bands across different tissues/lanes | alternative splicing gives rise to five annotated GAS6 isoforms of differing length |
💡Expected GAS6 appearanceExpect a predominant ~79 kDa band, N-glycosylated mature GAS6 close to its 74.9 kDa predicted mass after signal-peptide cleavage, plus a secondary ~55 kDa isoform-related band, best seen in secreted fractions rather than whole-cell lysate.
| Predicted mass (UniProt) | 74.9 kDa unmodified backbone sets the baseline migration before any modifications are considered |
| Signal peptide cleavage (residues 1-30) | removal of the signal peptide during secretion yields a mature protein slightly smaller than the full translated precursor |
| N-glycosylation at Asn420 | the added glycan raises apparent mass above the 74.9 kDa prediction, consistent with the ~79 kDa native band, and can broaden it into a smear |
| Alternative splicing (5 isoforms) | isoform-specific length differences can produce additional bands distinct from the main ~79 kDa species, such as the ~55 kDa band |
| Secreted subcellular localization | little protein accumulates intracellularly, so whole-cell lysate signal is much weaker than in serum or conditioned medium |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | GAS6 is secreted and does not accumulate intracellularly | probe conditioned media, serum, or plasma instead of, or in addition to, whole-cell lysate |
| Band higher than expected | N-glycosylation at Asn420 adds mass above the 74.9 kDa predicted backbone, producing the ~79 kDa native band | treat the sample with PNGase F and confirm the band shifts down toward the unmodified backbone |
| Band lower than expected | a shorter splice isoform among the five annotated GAS6 transcripts can migrate near 55 kDa | verify isoform identity with isoform-specific detection or mass spectrometry if the lower band is reproducible |
| Multiple bands | alternative splicing into five isoforms combined with variable glycosylation produces bands at both ~55 kDa and ~79 kDa | compare the pattern across tissues or cell lines and correlate with known isoform and glycosylation status |
| Broad smear instead of sharp band | heterogeneous occupancy of the single N-glycosylation site broadens the ~79 kDa band | run a longer resolving gel or deglycosylate the sample to sharpen the band |
| Weak or no signal | GAS6 is a comparatively low-abundance secreted factor outside enriched tissues | concentrate conditioned media or serum and increase sample loading or antibody incubation time |
Experiment evidence
Western blot images showing GAS6 band
A00608-1 · GAS6
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GAS6 Western blot
🧪For positive controls for GAS6 in Western blot, you can use recombinant human GAS6 protein or conditioned medium from a GAS6-secreting cell line, since none of the tissues in the supplied HPA data show clear positive detection.
Positive control: Recombinant GAS6 protein / conditioned medium
Negative control: Adipose tissue
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) to confirm equal loading.
⚠️Feasibility: GAS6 is secreted and shows no clearly positive tissue in the available HPA data, so whole-cell lysates likely give weak signal—favor conditioned medium or recombinant protein as the positive control and verify specificity with siRNA knockdown or a KO line.
HPA tissue expression evidence for GAS6
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GAS6 Western Blot Tips
Deeper troubleshooting and optimisation questions for GAS6, answered from its protein features.
Why does GAS6 run at 55 and 79 kDa instead of 75 kDa?
GAS6 carries N-glycosylation (1 site) and 16 disulfide bonds, which alter SDS-PAGE mobility. The 79 kDa band likely reflects the mature glycosylated secreted form; the 55 kDa band may be a proteolytically processed fragment lacking the Gla/EGF region. Confirm identity with reducing vs non-reducing gels.
Do GAS6 isoforms explain multiple Western blot bands?
UniProt lists 5 GAS6 isoforms from alternative splicing. Isoform-specific exon inclusion changes predicted mass, so antibodies targeting shared regions can detect multiple bands corresponding to different isoforms. Check the immunogen location against isoform sequences to determine which bands are expected for your antibody.
What induces GAS6 expression before Western blot analysis?
GAS6 (growth arrest-specific 6) is transcriptionally upregulated in growth-arrested, serum-starved, or quiescent cells, consistent with its growth-arrest-specific designation. Serum-starving cultures for 24-48h before lysis typically increases GAS6 signal compared with proliferating, serum-fed conditions.
How to optimize blocking for GAS6 detection?
Because GAS6 is a secreted glycoprotein with a Gla domain, use BSA rather than milk for blocking to avoid lectin-like carbohydrate interactions with milk glycoproteins that can raise background. Start with 5% BSA in TBST for 1h and titrate if background persists.
What transfer method to use for GAS6 Western blot?
GAS6 is a large (75 kDa), disulfide-rich secreted glycoprotein, so wet/tank transfer overnight at low voltage and 4C is recommended over rapid semi-dry transfer to ensure complete transfer of both the full-length and processed forms.
How should GAS6 be normalized in conditioned media?
Since GAS6 is secreted, not cytoplasmic, avoid housekeeping loading controls like GAPDH or actin, which are lysate-specific. Normalize conditioned-media samples to total secreted protein (e.g., Ponceau or BCA) or to cell number/volume instead.
What causes unexpected extra bands in GAS6 blots?
With 16 disulfide bonds, incomplete reduction can leave partially folded species running anomalously on non-reducing gels. Additionally, GAS6 forms a heterodimer or heterotetramer with AXL, so non-reducing, non-denaturing conditions may preserve higher molecular weight complexes beyond the monomeric band.
Boster reagents
Best GAS6 Western Blot Antibodies
BosterBio's GAS6 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-GAS 6/GAS6 Antibody Picoband®
Cat # A00608-1
Our recommended anti-GAS6 antibody is a top-performing, widely cited reagent for Western blot, thoroughly validated with clear specific banding and cross-checked against negative tissue controls and complementary detection methods for reliable, reproducible results.
Which to pick: Only one anti-GAS6 antibody is catalogued here, A00608-1, which includes an actual Western blot validation image showing specific GAS6 detection—making it the clear, and currently only, choice for your WB experiments.
Source: BosterBio GAS6 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14393.
- Human Protein Atlas. GAS6 tissue expression.