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SHBG · Western blot design guide
Design a Western Blot for SHBG
Real validated SHBG Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SHBG WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
SHBG 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 | ~43.8 kDa | |
| Observed band | 70 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Homodimer formation | |
| Regulation | LPS-induced | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated SHBG Western Blot Protocols
Literature-validated Western blot parameters for SHBG — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | BL41, Jurkat, A20, IP12-7 cell lysates |
| Gel % | 10–12% |
| Transfer | semi-dry blotter to nitrocellulose membrane |
| Membrane | nitrocellulose membrane (Bio-Rad) |
| Blocking | 5% BSA in TBS with 0.05% Tween-20, 1 hour |
| Primary incubation | overnight at 4°C |
| Secondary incubation | 1 hour |
| Wash | repeated washing with TBS-Tween buffer |
| Detection | enhanced chemiluminescence (ECL; Amersham International) |
Section 2
What Is the Expected SHBG Western Blot Band Size?
SHBG has a 43.8 kDa polypeptide backbone but runs at ~70 kDa on blots due to heavy N- and O-linked glycosylation, with a homodimer possible under non-reducing conditions.
| single band near 70 kDa | the mature, glycosylated SHBG monomer migrating well above its 43.8 kDa unmodified backbone |
| diffuse or smeared band around 65-75 kDa | heterogeneous glycoform occupancy across the three glycosylation sites (O-linked Thr36; N-linked Asn380, Asn396) |
| faint higher band near double the monomer mass under non-reducing conditions | incompletely reduced or intact SHBG homodimer stabilized by its disulfide bonds |
| little or no band in whole-cell lysate | SHBG is a secreted protein and accumulates in serum, conditioned media, or luminal fluid rather than inside cells |
| multiple closely spaced bands | detection of different SHBG splice isoforms rather than a single species |
💡Expected SHBG appearanceExpect a single glycosylated SHBG band at 70 kDa, well above the 43.8 kDa predicted backbone, driven by N- and O-glycosylation at three sites; a faint ~140 kDa band may reflect incomplete dimer reduction.
| Predicted mass (UniProt) | 43.8 kDa unmodified polypeptide backbone serves as the baseline before any modification is added |
| O-linked glycosylation at Thr36 | adds mass and heterogeneity, shifting and broadening the band above the predicted backbone |
| N-linked glycosylation at Asn380 and Asn396 | further increases apparent mass and contributes to the diffuse band consistent with the ~70 kDa observed size |
| Disulfide bonds (Cys193-Cys217, Cys362-Cys390) and homodimer formation | stabilize folding and can support a dimer species running near double the monomer mass if reduction is incomplete |
| Signal peptide (residues 1-29) | cleaved during secretion, so the mature secreted protein runs slightly smaller than the unprocessed precursor |
| Splice isoforms (5 named) | can generate additional bands of slightly different sizes depending on which isoform(s) are present |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SHBG is secreted and does not accumulate intracellularly | test serum, plasma, or conditioned media instead of whole-cell lysate |
| Band higher than expected | heavy N- and O-glycosylation shifts the 43.8 kDa backbone up to the ~70 kDa observed monomer, and incomplete reduction can allow the disulfide-stabilized homodimer to run near double that mass | compare against the empirical ~70 kDa band rather than predicted mass and ensure complete reduction with fresh reducing agent and full boiling |
| Broad smear instead of sharp band | heterogeneous occupancy and composition across the three glycosylation sites | treat the sample with a deglycosylation enzyme to collapse glycoforms into a single sharper band |
| Multiple bands | co-detection of different SHBG splice isoforms | check which isoform(s) the antibody epitope covers before interpreting extra bands as nonspecific |
| Weak or no signal | low abundance of secreted protein in the sampled compartment or epitope masking by glycosylation | increase loading, sample serum or secreted media, or deglycosylate to expose masked epitopes |
| Fragments below expected size | normal cleavage of the signal peptide during secretion removes residues 1-29 from the precursor | confirm the antibody targets mature secreted SHBG rather than the signal-peptide-containing precursor region |
Experiment evidence
Western blot images showing SHBG band
PB9326 · SHBG
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SHBG Western blot
🧪For positive controls for SHBG in Western blot, you can use testis tissue lysate, since SHBG is synthesized by Sertoli cells and secreted into the lumen of the seminiferous tubule.
Positive control: Testis (Sertoli cells)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: SHBG is a secreted protein, so whole-cell lysates may give weak signal, favoring conditioned medium or seminiferous fluid, and with no confirmed negative tissue available a siRNA knockdown or KO line is the more reliable negative control.
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 |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced SHBG Western Blot Tips
Deeper troubleshooting and optimisation questions for SHBG, answered from its protein features.
Why does SHBG run near 70 kDa instead of predicted 43.8 kDa?
SHBG carries three predicted N-glycosylation sites; the added glycan mass typically shifts the apparent molecular weight upward, explaining an observed band near 70 kDa despite a 43.8 kDa unmodified core sequence. Confirm identity with a glycosylation-sensitive control or PNGase F digestion if the shift needs verification.
Which of the five SHBG isoforms will my blot detect?
UniProt lists five annotated SHBG isoforms arising from alternative splicing. Standard antibodies raised against shared regions typically detect multiple isoforms as one or more close bands; if isoform-specific resolution is needed, use antibodies targeting unique spliced exons and run high-percentage gels to separate closely sized variants.
Does glycosylation affect SHBG antibody epitope recognition?
SHBG has three glycosylation sites but no reported phosphorylation or other modified residues. Some antibodies raised against non-glycosylated recombinant antigen may show reduced affinity for the native glycosylated protein; if signal is weak, consider antibodies validated against native serum-purified SHBG rather than bacterially expressed antigen.
How should blocking be optimized for glycosylated SHBG detection?
Because SHBG is a glycoprotein, milk-based blockers containing glycan-binding lectins can increase background from non-specific lectin-glycan interactions. Use BSA-based blocking buffer instead of milk to reduce noise, particularly when probing serum samples where SHBG circulates alongside other glycosylated proteins.
What transfer method to use for SHBG Western blot?
At a predicted 43.8 kDa (observed near 70 kDa with glycosylation), standard wet or semi-dry transfer at low-to-moderate voltage is sufficient. Because SHBG forms a homodimer stabilized partly by disulfide bonds, ensure samples are fully reduced and denatured before loading so transfer targets the monomeric, glycosylated form rather than partially resistant dimers.
Should SHBG samples be run reduced to avoid dimer artifacts?
SHBG is a homodimer with two disulfide bonds per evidence. Non-reducing conditions can preserve disulfide-linked dimers, roughly doubling apparent mass and complicating quantitation against a monomer standard curve. Always use reducing sample buffer (DTT/beta-mercaptoethanol) and confirm complete reduction so quantitation reflects monomeric SHBG consistently across samples.
What could cause an unexpected higher-mass SHBG band?
Incomplete reduction of the two disulfide bonds can leave dimeric or partially disulfide-linked SHBG species migrating above the ~70 kDa glycosylated monomer. Variable glycan occupancy across the three glycosylation sites can also produce a diffuse or doublet band pattern; treat samples with stronger reducing agent and consistent glycosylation status to resolve this.
Boster reagents
Best SHBG Western Blot Antibodies
BosterBio's SHBG 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-Sex Hormone Binding Globulin/SHBG Antibody Picoband®
Cat # PB9326
Our recommended anti-SHBG antibody is a best-performing, extensively cited reagent, thoroughly validated by Western blot and orthogonally cross-validated against negative tissue and complementary detection methods to ensure specific, reproducible SHBG detection across sample types.
Which to pick: Only one Boster SHBG antibody is listed: PB9326 (Picoband), backed by an actual Western blot image showing a ~44 kDa band in HeLa and COLO320 whole-cell lysates at 0.5 ug/ml—making it the default, validated choice.
Source: BosterBio SHBG gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P04278.
- Human Protein Atlas. SHBG tissue expression.