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- Table of Contents
HMGA2 / High mobility group protein HMGI-C · Western blot design guide
Design a Western Blot for HMGA2
Real validated HMGA2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HMGA2 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
HMGA2 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 | ~11.8 kDa | |
| Observed band | ~18–20 kDa | |
| Gel | 10% (catalog A00436-3) | |
| Positive control | Testis (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 6 isoform(s) |
Section 1
Real Curated HMGA2 Western Blot Protocols
The A00436-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human HepG2, rat C6 (catalog A00436-3) |
| Gel % | 10% (catalog A00436-3) |
| Load | 30 ug; reducing conditions (catalog A00436-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00436-3) |
| Membrane | nitrocellulose membrane (catalog A00436-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00436-3) |
| Primary antibody | A00436-3 · 1:1000 (catalog A00436-3) |
| Primary incubation | overnight at 4°C (catalog A00436-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00436-3) |
| Secondary incubation | 1.5 hour at RT (catalog A00436-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00436-3) |
| Detection | ECL (catalog A00436-3) |
Section 2
What Is the Expected HMGA2 Western Blot Band Size?
HMGA2 is predicted at 11.8 kDa, while antibody QC reports ~18–20 kDa; the cause of the difference is not established.
| Band at ~18–20 kDa | Empirical HMGA2 band reported in whole-cell lysates; the difference from predicted mass is unexplained. |
| Band near 11.8 kDa | Near the predicted mass of HMGA2; confirm its identity with antibody controls. |
| Several bands | Isoforms 1–6 are annotated, but their masses and separation on a blot are unknown. |
| Close doublet | Phosphorylation could affect migration, but a visible doublet has not been established. |
💡Expected HMGA2 appearanceHMGA2 has a predicted mass of 11.8 kDa, while antibody QC reports a band at ~18–20 kDa; the cause of this difference is unknown, so confirm band identity with appropriate controls.
| Predicted HMGA2 mass | 11.8 kDa is the sequence-based reference, not the reported apparent band size of ~18–20 kDa. |
| Splice isoforms 1–6 | May differ in size, but their individual masses and migration are not supplied. |
| Phosphorylation at Thr40 and Ser44 | Could affect apparent migration; no visible shift is established. |
| Phosphorylation at Ser101 and Ser105 | Could affect apparent migration; no visible shift is established. |
| N-acetylserine at residue 2 | Adds a modification, with no demonstrated effect on band position. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear HMGA2 may be poorly recovered or absent from the tested sample. | Check nuclear extraction and include an HMGA2-positive lysate. |
| Band higher than expected | The reported ~18–20 kDa band exceeds the 11.8 kDa prediction for an unestablished reason. | Compare with the antibody QC band and verify identity by HMGA2 depletion. |
| Band lower than expected | An alternate isoform is possible, but isoform band sizes are unknown. | Confirm with HMGA2 depletion and an antibody against another epitope. |
| Multiple bands | Isoforms or phosphorylation are possible contributors, but distinct bands are unverified. | Test band identity by HMGA2 depletion and assess phosphorylation with phosphatase treatment. |
| Weak or no signal | Low HMGA2 abundance or poor recovery of this nuclear protein. | Use a positive control and check nuclear protein recovery. |
Experiment evidence
Western blot images showing HMGA2 band
A00436-3 · HMGA2
A00436-1 · HMGA2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HMGA2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HMGA2 in Western blot, you can use testis tissue, where HPA reports high expression.
Positive control: Testis (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: HMGA2 is nuclear, so nuclear extracts may improve detection.
HPA tissue expression evidence for HMGA2
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 |
|---|---|---|---|---|
| Testis | preleptotene spermatocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | Medium | 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 → |
| Seminal vesicle | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Breast | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced HMGA2 Western Blot Tips
Deeper troubleshooting and optimisation questions for HMGA2, answered from its protein features.
How should HMGA2 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.
Can HMGA2 isoforms produce different bands?
Isoforms · UniProt lists six isoforms. In isoforms 2–6, the canonical sequence at residues 84–109 is replaced by sequences of different lengths. Compare the antibody’s target region with each isoform before interpreting multiple bands; the sequence differences do not guarantee distinct visible bands.
The UniProt positions refer to the canonical sequence. Isoforms 2–6 replace canonical residues 84–109, so canonical phosphoserines 101 and 105 cannot be assumed to occur at the same positions in those isoforms. Check each variant sequence before comparing site-specific results.
The UniProt positions refer to the canonical sequence. Isoforms 2–6 replace canonical residues 84–109, so canonical phosphoserines 101 and 105 cannot be assumed to occur at the same positions in those isoforms. Check each variant sequence before comparing site-specific results.
Which HMGA2 modifications matter when interpreting bands?
PTM · Using canonical UniProt numbering, HMGA2 has N-acetylserine at residue 2, phosphothreonine at 40, and phosphoserine at 44, 101, and 105. These sites can guide follow-up experiments, but their presence does not establish a visible shift or explain the ~18–20 kDa band.
Does this guide establish induction of HMGA2?
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 HMGA2 Western blot?
Transfer · Canonical HMGA2 is small at 11.8 kDa. Choose and check transfer conditions for retention of a small protein, and inspect the membrane and gel after transfer. The supplied UniProt features do not specify a transfer method; optimize it empirically for the band being measured.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00436-3 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 HMGA2 band intensity be quantified?
Quantitation · Quantify the same resolved band across samples with consistent sample preparation and normalization. HMGA2 is nuclear, so keep the sampled fraction consistent. Because UniProt lists six isoforms, establish which bands the antibody detects before combining their intensities.
Why might HMGA2 appear at 18–20 kDa instead of 11.8 kDa?
Interpretation · The supplied apparent band is ~18–20 kDa, while canonical HMGA2 has a predicted mass of 11.8 kDa. UniProt lists six isoforms and several modifications, but those features alone do not establish the cause of the difference. Verify band identity before assigning it to HMGA2.
Check whether the antibody’s target region is present in the relevant isoforms: isoforms 2–6 replace canonical residues 84–109. Compare bands with the 11.8 kDa predicted canonical mass and the supplied ~18–20 kDa apparent band, but do not assign identity from migration or listed modifications alone.
UniProt places HMGA2 in the nucleus. Use a consistent preparation that includes nuclear protein when comparing samples, and keep the preparation method the same for band intensity measurements.
Check whether the antibody’s target region is present in the relevant isoforms: isoforms 2–6 replace canonical residues 84–109. Compare bands with the 11.8 kDa predicted canonical mass and the supplied ~18–20 kDa apparent band, but do not assign identity from migration or listed modifications alone.
UniProt places HMGA2 in the nucleus. Use a consistent preparation that includes nuclear protein when comparing samples, and keep the preparation method the same for band intensity measurements.
Boster reagents
HMGA2 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-HMGA2 Antibody
Cat # A00436-3
Real WB data
Anti-HMGA2 Antibody (C-term)
Cat # A00436-1
Two anti-HMGA2 antibodies have Western blot images. A00436-3 was tested in human, rat, and mouse cell lysates, with a reported band at 18–20 kDa versus the expected 12 kDa. A00436-1 was shown in human HepG2 lysate; its caption gives no band size.
Which to pick: For human, rat, or mouse lysates, consider A00436-3 based on the pictured A549, HepG2, C6, and NIH/3T3 samples, while accounting for the reported band-size discrepancy. For human HepG2 lysate, A00436-1 also has a Western blot image. Neither image establishes performance in every sample.
Source: BosterBio HMGA2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P52926.
- Human Protein Atlas. HMGA2 tissue expression.
- PMC5437003 — target-verified WB comparison
- PMC3629159 — target-verified WB comparison
- PMC6505466 — target-verified WB comparison