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- Table of Contents
FST · Western blot design guide
Design a Western Blot for FST
Real validated FST Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FST WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
FST 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 | ~38 kDa | |
| Observed band | ~40 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 + Cleaved | |
| Caveat | Glycosylation increases size | |
| Regulation | Myogenesis | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated FST Western Blot Protocols
Literature-validated Western blot parameters for FST — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | mouse kidney , Lane 2: human MCF-7 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-Follistatin antigen affinity purified polyclonal antibody (Catalog # A00972-1) at 0.5 ug/mL overnight at 4 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 1:10000 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 Follistatin at approximately 40KD. The expected band size for Follistatin is at 38KD |
| Gel % | 10–12% |
| Load | 50ug |
| 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 |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 40 kDa |
Section 2
What Is the Expected FST Western Blot Band Size?
FST has a 38 kDa predicted backbone but runs at approximately 40 kDa due to N-glycosylation at Asn124 and Asn288 following signal peptide cleavage.
| Single band at ~40 kDa | Mature, signal-peptide-cleaved follistatin carrying N-glycans at Asn124 and Asn288, running above its 38 kDa predicted mass |
| Diffuse or slightly smeared band around 38-42 kDa | Heterogeneous glycoform occupancy across the two N-linked glycosylation sites |
| Faint band just below the mature ~40 kDa band | Residual unglycosylated or signal-peptide-uncleaved precursor form |
| Two closely spaced bands | Co-expression of the two annotated splice isoforms (isoform 1 and isoform 2) |
| Little or no band in whole-cell lysate | Follistatin is actively secreted, so intracellular pools can be minimal |
💡Expected FST appearanceFollistatin typically appears as a band at approximately 40 kDa, slightly above its 38 kDa predicted mass, reflecting cleavage of the N-terminal signal peptide and N-glycosylation at Asn124 and Asn288 on the secreted mature protein.
| Predicted mass (UniProt) | 38 kDa unmodified backbone sets the baseline expectation before processing and modification |
| Signal peptide cleavage (residues 1-29) | Removal of the N-terminal signal peptide produces the mature secreted form, smaller than the unprocessed precursor |
| N-glycosylation at Asn124 and Asn288 | Two N-linked glycans add mass, shifting the mature band up toward the ~40 kDa observed size and potentially broadening it |
| Alternative splicing (isoforms 1 and 2) | The two named isoforms can migrate at slightly different apparent sizes, producing an extra band relative to the canonical form |
| Secreted subcellular localization | Signal is often stronger in conditioned media or serum/plasma than in whole-cell lysate |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Follistatin is a secreted protein with limited intracellular retention | Test conditioned media, serum, or plasma alongside or instead of whole-cell lysate |
| Band higher than expected | N-glycosylation at Asn124 and Asn288 adds mass above the 38 kDa predicted backbone | Treat the ~40 kDa empirical band as the expected size rather than the unmodified predicted mass, and confirm with PNGase F treatment as a lower-bound control |
| Broad smear instead of sharp band | Heterogeneous glycosylation across the two N-linked sites produces a range of glycoforms | Run a longer resolving gel or deglycosylate with PNGase F to collapse the smear into a single sharp band |
| Multiple bands | The two annotated splice isoforms may be co-expressed in the same sample | Verify isoform identity with mass comparison or an isoform-specific antibody epitope |
| Weak or no signal | Low secretion output or dilution of the secreted antigen in conditioned media | Concentrate conditioned media or increase sample loading and exposure time before repeating |
Experiment evidence
Western blot images showing FST band
A00972-1 · FST
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FST Western blot
🧪For positive controls for FST in Western blot, you can use recombinant Follistatin protein or conditioned medium from an FST-secreting cell line, since none of the HPA-profiled tissues (adipose tissue, adrenal gland, appendix, bone marrow) showed detectable expression.
Positive control: Recombinant FST protein / conditioned medium
Negative control: Adipose tissue
Loading controls: Run GAPDH and β-actin blots alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: FST is secreted, so whole-cell lysates from the HPA-profiled tissues show little to no signal, making conditioned medium or recombinant protein a more feasible positive control than any tissue lysate.
HPA tissue expression evidence for FST
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 | lymphoid tissue | 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 | lymphoid tissue | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FST Western Blot Tips
Deeper troubleshooting and optimisation questions for FST, answered from its protein features.
Why does FST migrate near 40 kDa instead of 38 kDa?
UniProt lists 2 N-glycosylation sites on follistatin. Added carbohydrate mass typically shifts secreted glycoproteins slightly above their predicted molecular weight, so an observed band around 40 kDa versus a predicted 38 kDa is consistent with normal glycosylation and not a specificity concern.
Should I expect more than one Follistatin isoform on blot?
UniProt annotates 2 isoforms (1 and 2) generated by alternative splicing. Confirm your antibody's immunogen maps to a region shared by both isoforms if you want to detect total follistatin, or check the datasheet if you need isoform-selective detection.
Does activin signaling influence Follistatin levels detected?
Follistatin directly binds activin A, GDF11, and myostatin as an antagonist. Because this interaction is part of a negative feedback loop, cells or tissues with active TGF-beta/activin signaling may show altered follistatin abundance, which is worth noting when comparing treatment conditions.
How should blocking be optimized given follistatin's disulfide bonding?
Follistatin has 18 annotated disulfide bonds, making it a highly cross-linked, compact glycoprotein. Ensure sample buffer contains a reducing agent (DTT or beta-mercaptoethanol) and full heat denaturation before loading; standard 5% milk or BSA blocking is otherwise sufficient since no other PTMs are annotated.
What transfer method to use for FST Western blot?
Follistatin is a small (~38 kDa), disulfide-rich secreted glycoprotein, so standard wet or semi-dry transfer to PVDF membrane works well. Use lower voltage/shorter transfer times appropriate for low-molecular-weight proteins to avoid blow-through, and confirm efficient transfer of the compact, highly cross-linked structure.
What loading control is appropriate for quantitating secreted Follistatin?
UniProt annotates follistatin as Secreted, so if analyzing conditioned media or serum, avoid cytosolic housekeeping proteins like GAPDH or actin as normalizers. Use a total protein stain (e.g., Ponceau S) or a validated secreted-protein loading control instead.
What could explain extra or higher molecular weight bands?
With 18 disulfide bonds, incomplete reduction can leave misfolded or aggregated species running higher than expected. Follistatin also interacts with activin A, GDF11, and myostatin; residual complexes with these binding partners under non-reducing conditions could appear as additional higher-molecular-weight bands.
Could nuclear Follistatin localization affect band interpretation?
UniProt notes follistatin localizes to both the secreted extracellular space and the nucleolus. If probing whole-cell lysate versus conditioned media, band intensity may differ by compartment; consider subcellular fractionation if nuclear versus secreted pools need to be distinguished.
Boster reagents
Best FST Western Blot Antibodies
BosterBio's FST 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-Follistatin/FST Antibody Picoband®
Cat # A00972-1
Our recommended anti-Follistatin antibody is a top-performing, extensively cited reagent, rigorously validated by Western blot and cross-checked against negative tissue and complementary detection methods to ensure specificity and reliable results for your FST studies.
Which to pick: Only one Boster FST antibody is catalogued here, A00972-1, which includes an actual Western blot validation image—making it the clear choice for reliable, image-confirmed detection of Follistatin in your samples.
Source: BosterBio FST gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P19883.
- Human Protein Atlas. FST tissue expression.