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- Table of Contents
HLA-G / HLA class I histocompatibility antigen, alpha chain G · Western blot design guide
Design a Western Blot for HLA-G
Real validated HLA-G Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HLA-G 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
HLA-G 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 | ~38.2 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 7 isoform(s) |
Section 1
Real Curated HLA-G Western Blot Protocols
The M01235 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Jurkat cell lysate (catalog M01235) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M01235; 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 HLA-G Western Blot Band Size?
HLA-G has a predicted 38.2 kDa precursor; its features could affect migration, but no empirical band size or visible shift is established.
| Band near 38.2 kDa | compatible with the predicted HLA-G precursor mass; identity requires controls |
| Band below 38.2 kDa | may reflect signal peptide cleavage or a shorter isoform |
| Band above 38.2 kDa | may reflect N-linked glycosylation at Asn110 |
| Band near twice the monomer size under nonreducing conditions | may reflect an interchain disulfide-linked HLA-G dimer |
| Several bands at different sizes | may reflect isoforms 1 through 7, if their migration differs |
💡Expected HLA-G appearanceUniProt predicts a 38.2 kDa precursor, but no empirical band size is supplied; signal peptide cleavage, Asn110 glycosylation, isoforms, and disulfide-linked dimers may alter migration, so confirm identity with controls.
| UniProt predicted precursor mass | 38.2 kDa provides the unmodified precursor reference |
| N-linked glycosylation at Asn110 | may increase apparent mass; the size of any shift is unknown |
| Interchain disulfides at Cys66 and Cys171 | may yield a band near twice the monomer size without complete reduction |
| Isoforms 1, 2, 3, 4, 5, 6, and 7 | may differ in size; their individual masses and migration are unspecified |
| Signal peptide at residues 1–24 | cleavage yields a mature chain smaller than the precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | membrane-localized isoform 1 may be underrepresented | check a membrane-enriched fraction and a positive control |
| Band higher than expected | incomplete reduction may retain an interchain disulfide-linked dimer | compare reducing and nonreducing samples |
| Band lower than expected | signal peptide cleavage or a shorter isoform may reduce size | check antibody epitope coverage and compare an HLA-G positive control |
| Broad smear instead of sharp band | variable glycosylation at Asn110 is possible but unproven | compare matched samples before and after N-glycan removal |
| Multiple bands | isoforms or differing modification states may contribute | compare reducing conditions and use HLA-G depletion to assess band identity |
Experiment evidence
Western blot images showing HLA-G band
M01235 · HLA-G
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HLA-G Western blot
🧪For positive controls for HLA-G in Western blot, you can use an HPA-confirmed positive sample, but none is identified in the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside samples.
⚠️Feasibility: Secreted HLA-G may give weak whole-cell lysate signal; consider conditioned medium.
HPA tissue expression evidence for HLA-G
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced HLA-G Western Blot Tips
Deeper troubleshooting and optimisation questions for HLA-G, answered from its protein features.
How should HLA-G 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.
Which HLA-G isoforms could produce different bands?
Isoforms · Seven isoforms are listed. Isoforms 2 and 6 lack residues 115–206; isoform 3 lacks 115–298; isoform 4 lacks 207–298; and isoform 7 lacks 117–338. Isoforms 5 and 6 have a different sequence at 299–338. Check whether the antibody recognizes a region retained in the isoform being measured.
Choose the band or bands to quantify based on which isoforms the antibody can recognize. Several isoforms lack large parts of the canonical sequence, so signal from one band may not represent all seven isoforms. Keep the same band-selection rule across samples.
UniProt places isoform 1 at the cell membrane, endoplasmic reticulum membrane, and early endosome membrane. Include a fraction containing these membranes when assessing isoform 1; a fraction that excludes them could give a weak signal.
Choose the band or bands to quantify based on which isoforms the antibody can recognize. Several isoforms lack large parts of the canonical sequence, so signal from one band may not represent all seven isoforms. Keep the same band-selection rule across samples.
UniProt places isoform 1 at the cell membrane, endoplasmic reticulum membrane, and early endosome membrane. Include a fraction containing these membranes when assessing isoform 1; a fraction that excludes them could give a weak signal.
Where is the listed HLA-G glycosylation site?
PTM · UniProt lists one N-linked glycosylation site at asparagine 110. Use that UniProt coordinate when comparing annotations; numbering in papers or antibody materials may use a different convention. The listed site alone does not establish a visible band shift.
Does this guide establish induction of HLA-G?
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.
How should transfer be checked for HLA-G?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M01235 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 HLA-G be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might HLA-G migrate differently from its predicted 38.2 kDa?
Interpretation · The 38.2 kDa prediction refers to the 338-residue sequence, which includes a signal peptide at residues 1–24. HLA-G also has an N-linked glycosylation site at residue 110. These features affect the mature protein, but the supplied evidence does not establish an apparent band size or explain a particular shift.
Compare each band's antibody epitope with the listed alternative sequences before assigning it to an isoform. Also consider the signal peptide at 1–24 and the glycosylation site at 110. No observed band size is supplied, so these features cannot identify an unexpected band by size alone.
UniProt lists four disulfide bonds. Record whether samples were reduced and use consistent conditions when comparing lanes, since disrupting these bonds can affect protein conformation. The supplied features do not predict a specific band change under reducing conditions.
Compare each band's antibody epitope with the listed alternative sequences before assigning it to an isoform. Also consider the signal peptide at 1–24 and the glycosylation site at 110. No observed band size is supplied, so these features cannot identify an unexpected band by size alone.
UniProt lists four disulfide bonds. Record whether samples were reduced and use consistent conditions when comparing lanes, since disrupting these bonds can affect protein conformation. The supplied features do not predict a specific band change under reducing conditions.
Boster reagents
HLA-G 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-HLA G Rabbit Monoclonal Antibody
Cat # M01235
The catalog reports a rabbit monoclonal anti-HLA-G antibody with stated human reactivity. Its Western blot image shows HLA-G expression in Jurkat cell lysate; the supplied evidence does not establish performance in other samples.
Which to pick: M01235 is the only listed option. It has a Western blot image using Jurkat cell lysate and stated human reactivity; assess suitability for your sample and conditions.
Source: BosterBio HLA-G gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P17693.
- Human Protein Atlas. HLA-G tissue expression.
- PMC6197483 — target-verified WB comparison
- PMC5814230 — target-verified WB comparison
- PMC3385011 — target-verified WB comparison