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- Table of Contents
TG / Thyroglobulin · Western blot design guide
Design a Western Blot for TG
Real validated TG Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TG 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
TG 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 | ~304.8 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Positive control | Thyroid gland (IHC candidate; verify WB) | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Extensive glycosylation | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated TG Western Blot Protocols
The M00359-5 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Human thyroid lysate (catalog M00359-5) |
| Gel % | 4–12% gradient (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 | M00359-5; 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 TG Western Blot Band Size?
TG has a 304.8 kDa predicted backbone; extensive N-linked glycosylation raises and may broaden its monomer band, while dimers or oligomers can run higher.
| High band above 304.8 kDa, possibly broad | N-linked glycans increase apparent monomer size |
| Band near twice the monomer size under nonreducing conditions | TG homodimers or disulfide-linked oligomers persist |
| Band slightly below the full-length precursor | The signal peptide has been cleaved |
| Little or no band in whole-cell lysate | TG is secreted into the thyroid follicle lumen |
| Several bands at different sizes | Isoforms 1 and 2 may contribute distinct bands |
💡Expected TG appearanceExpect a glycosylated TG monomer above its 304.8 kDa predicted backbone mass, with possible broadening; nonreducing conditions may reveal dimers or higher oligomers.
| 304.8 kDa predicted backbone | Provides the full-length mass before glycosylation and signal-peptide cleavage |
| N-linked glycosylation at Asn76 and Asn110 | Raises and may broaden apparent monomer size |
| N-linked glycosylation at Asn198 and Asn484 | Adds glycan mass and may vary between molecules |
| Signal peptide at residues 1–19 | Cleavage makes mature TG slightly smaller than the precursor |
| Homodimer and disulfide-linked oligomers | Can produce bands around twice the monomer size or higher under nonreducing conditions |
| Splice isoforms 1 and 2 | May produce bands of differing sizes |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | TG is secreted into the thyroid follicle lumen | Check follicular lumen or secreted material alongside thyroid lysate |
| Band higher than expected | N-linked glycans or persistent homodimers and oligomers | Compare reducing and nonreducing samples; assess glycosylation |
| Band lower than expected | Signal-peptide cleavage or a smaller splice isoform | Compare precursor and mature forms and check isoform coverage |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation | Compare untreated and deglycosylated samples |
| Multiple bands | Isoforms 1 and 2 or persistent oligomers | Compare reducing conditions and check which isoforms the antibody recognizes |
| Fragments below expected size | Proteolysis of TG | Handle samples promptly with protease inhibitors |
Experiment evidence
Western blot images showing TG band
M00359-5 · TG
M00359-3 · TG
M00359-4 · TG
M00359-6 · TG
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TG Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TG in Western blot, you can use thyroid gland tissue, where HPA reports high expression.
Positive control: Thyroid gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: TG is secreted into the thyroid follicle lumen, so thyroid tissue that includes follicular contents may give a stronger signal than isolated cells.
HPA tissue expression evidence for TG
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 |
|---|---|---|---|---|
| Thyroid gland | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-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 TG Western Blot Tips
Deeper troubleshooting and optimisation questions for TG, answered from its protein features.
How should TG 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.
Could TG isoforms produce different bands?
Isoforms · UniProt lists two TG isoforms, 1 and 2, and alternative splicing. Distinct bands could reflect these isoforms, but the supplied features do not give their individual masses; band position alone cannot assign an isoform.
Could TG modifications explain multiple bands?
PTM · TG has 21 glycosylation sites, 36 modified residues, and iodination and sulfation annotations. Differences in modification may contribute to band heterogeneity. Avoid assigning a particular modification from migration alone.
Does this guide establish induction of TG?
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 TG Western blot?
Transfer · TG is a large protein with a predicted mass of 304.8 kDa and can form higher-order assemblies. Check that your chosen transfer conditions move high-mass TG out of the gel and retain it on the membrane; inspect both gel and membrane when signal is weak.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00359-5 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.
Which sample location matters when quantifying TG?
Quantitation · TG is secreted into the thyroid follicle lumen and localizes to colloid globules. Quantify comparable sample fractions across conditions; changes in a cellular fraction alone may reflect secretion or localization rather than total TG abundance.
Why might TG migrate above its predicted 304.8 kDa mass?
Interpretation · TG has 21 annotated glycosylation sites and 36 modified residues. These modifications can change apparent migration, so use 304.8 kDa as a sequence-based reference rather than an exact band position.
TG has a signal peptide at residues 1–19 and is secreted into the thyroid follicle lumen. Its mature secreted form lacks that signal peptide, so compare bands with the processed protein as well as the 304.8 kDa precursor reference.
TG forms homodimers through its ChEL region and disulfide-linked homooligomers. High-mass bands may represent these assemblies. Compare reducing and nonreducing conditions before interpreting them as separate proteins.
TG has a signal peptide at residues 1–19 and is secreted into the thyroid follicle lumen. Its mature secreted form lacks that signal peptide, so compare bands with the processed protein as well as the 304.8 kDa precursor reference.
TG forms homodimers through its ChEL region and disulfide-linked homooligomers. High-mass bands may represent these assemblies. Compare reducing and nonreducing conditions before interpreting them as separate proteins.
Boster reagents
TG 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
Anti-Thyroglobulin Rabbit Monoclonal Antibody
Cat # M00359-5
Real WB data
Anti-Thyroglobulin TG Monoclonal Antibody
Cat # M00359-3
Real WB data
Anti-Thyroglobulin TG Monoclonal Antibody
Cat # M00359-4
Real WB data
Anti-Thyroglobulin Rabbit Monoclonal Antibody
Cat # M00359-6
Four the supplier anti-TG antibodies are listed with Western blot images: three using human thyroid lysate and one using mouse thyroid lysate. The supplied evidence is limited to those image descriptions; no publication evidence is provided.
Which to pick: For human thyroid samples, consider M00359-5, M00359-4, or M00359-6, each with a human thyroid lysate WB image. For mouse thyroid samples, M00359-3 has the mouse thyroid lysate WB image. The supplied evidence does not distinguish among the human options.
Source: BosterBio TG gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P01266.
- Human Protein Atlas. TG tissue expression.
- PMC4917686 — target-verified WB comparison
- PMC7950113 — target-verified WB comparison