This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
HLA-F / HLA class I histocompatibility antigen, alpha chain F · Western blot design guide
Design a Western Blot for HLA-F
Source-linked HLA-F Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HLA-F 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-F Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~39.1 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 | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked HLA-F Western Blot Protocol Options
The M04798-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | JAR cell lysate (catalog M04798-1) |
| 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 | M04798-1; 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-F Western Blot Band Size?
HLA-F has a predicted 39.1 kDa precursor; its signal peptide, Asn107 glycosylation, and isoforms could affect migration, but no band position is demonstrated.
| Band near 39.1 kDa | Consistent with the predicted HLA-F precursor mass; identity requires controls |
| Band below 39.1 kDa | Could reflect removal of the 1–21 signal peptide; mature mass is not supplied |
| Band with altered mobility | Could reflect N-linked glycosylation at Asn107; the site alone does not establish a visible shift |
| Several bands at different positions | Could involve isoforms 1, 2, and 3; their migration differences are not established |
💡Expected HLA-F appearanceUniProt predicts a 39.1 kDa HLA-F precursor, while signal-peptide cleavage, N-linked glycosylation, and isoforms may affect migration; no empirical band size is supplied, so confirm band identity with appropriate controls.
| UniProt predicted precursor mass | 39.1 kDa provides a sequence-based reference, not a validated blot position |
| N-linked glycosylation at Asn107 | May alter apparent size, but the site does not establish a measurable shift |
| Signal peptide at residues 1–21 | Cleavage may make mature HLA-F smaller than the precursor; mature mass is not supplied |
| Isoforms 1, 2, and 3 | May differ in size; their relative masses and resolvable migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane HLA-F may be poorly recovered during extraction | Check membrane extraction and include a known HLA-F-positive control |
| Band higher than expected | N-linked glycosylation at Asn107 may affect migration | Compare matched samples before and after N-glycan removal and verify band identity |
| Band lower than expected | The 1–21 signal peptide may be removed during maturation | Check antibody recognition and compare with a known HLA-F-positive sample |
| Multiple bands | Isoforms 1, 2, and 3 are listed, but distinct bands are unproven | Check isoform expression and confirm candidate bands with an independent HLA-F control |
| Broad smear instead of sharp band | Variable N-linked glycosylation is possible but not demonstrated | Test N-glycan removal and verify HLA-F specificity with a control |
Experiment evidence
Western blot images showing HLA-F band
M04798-1 · HLA-F
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HLA-F Western blot
🧪For positive controls for HLA-F in Western blot, you can use a verified HLA-F-positive sample; the supplied HPA data identify no tissue or cell line.
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 alongside the samples.
⚠️Feasibility: HPA provides no expression data to select tissue controls, and HLA-F is a membrane protein.
HPA tissue expression evidence for HLA-F
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-F Western Blot Tips
Deeper troubleshooting and optimisation questions for HLA-F, answered from its protein features.
How should HLA-F 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.
How could HLA-F isoforms affect band interpretation?
Isoforms · UniProt lists three isoforms. Isoform 2 lacks residues 204..295, while isoform 3 has an alternative sequence beginning at residue 346. An antibody targeting residues 204..295 may miss isoform 2. Check the antibody epitope before assigning different bands to isoforms.
Which HLA-F glycosylation site matters for Western blot analysis?
PTM · UniProt lists one N-linked glycosylation site, Asn107, using full-length sequence numbering. Account for this site when comparing apparent and predicted mass, but do not attribute a particular band shift to glycosylation without experimental evidence.
Does this guide establish induction of HLA-F?
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-F?
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 M04798-1 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.
What should be controlled when quantifying HLA-F bands?
Quantitation · HLA-F is a single-pass membrane protein found at the cell membrane and on early endosome and lysosome membranes. Use consistent sample preparation and compare the same band across samples. Check antibody epitope coverage if the assay may detect more than one of the three listed isoforms.
Why might HLA-F migrate differently from its predicted 39.1 kDa?
Interpretation · The 39.1 kDa prediction is for the canonical sequence, which includes a signal peptide at residues 1..21. Signal peptide removal and N-linked glycosylation at Asn107 may affect the apparent mass. No observed band size is supplied, and these features alone do not establish a visible shift.
UniProt lists two disulfide bonds. Compare reducing conditions consistently across samples when assessing migration; the feature record does not establish whether reduction produces a visible band change.
Consider the signal peptide at residues 1..21, N-linked glycosylation at Asn107, and the three isoforms when evaluating band sizes. HLA-F also forms a heterotrimer with B2M and a self-peptide. These features suggest possibilities to investigate, but none identifies an unexpected band on its own.
UniProt lists two disulfide bonds. Compare reducing conditions consistently across samples when assessing migration; the feature record does not establish whether reduction produces a visible band change.
Consider the signal peptide at residues 1..21, N-linked glycosylation at Asn107, and the three isoforms when evaluating band sizes. HLA-F also forms a heterotrimer with B2M and a self-peptide. These features suggest possibilities to investigate, but none identifies an unexpected band on its own.
Boster reagents
HLA-F 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 F Rabbit Monoclonal Antibody
Cat # M04798-1
The listed anti-HLA-F rabbit monoclonal antibody is catalogued as human-reactive and has a Western blot image from JAR cell lysate. The supplied evidence documents this tested context; broader sample performance is not established.
Which to pick: M04798-1 is the only listed option and the only one with a Western blot image. Its documented example uses JAR cell lysate; assess suitability for your sample and conditions.
Source: BosterBio HLA-F gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P30511.
- Human Protein Atlas. HLA-F tissue expression.
- PMC4992421 — target-verified WB comparison
- PMC3867582 — target-verified WB comparison
- PMC7519664 — target-verified WB comparison