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- Table of Contents
HLA-B / HLA class I histocompatibility antigen, B alpha chain · Western blot design guide
Design a Western Blot for HLA-B
Real validated HLA-B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HLA-B 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-B 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 | ~40.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (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 | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated HLA-B Western Blot Protocols
The M00186-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Ramos cell lysate (catalog M00186-2) |
| 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 | M00186-2; 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-B Western Blot Band Size?
UniProt predicts a 40.5 kDa precursor; signal-peptide cleavage and Asn110 glycosylation may affect migration, but no empirical band or visible shift is established.
| Band near 40.5 kDa | Consistent with the predicted HLA-B alpha-chain precursor; confirm band identity with controls. |
| Band below 40.5 kDa | Could reflect cleavage of the signal peptide at residues 1–24. |
| Band above the predicted size | N-linked glycosylation at Asn110 could affect migration; the size of any shift is unknown. |
| Two bands near the expected size | Could reflect precursor and signal-peptide-cleaved forms; their separation is unverified. |
💡Expected HLA-B appearanceUniProt predicts a 40.5 kDa HLA-B alpha-chain precursor; signal-peptide cleavage and Asn110 glycosylation may affect migration, but no empirical band size is supplied, so confirm identity with appropriate controls.
| UniProt predicted precursor mass | Sets a 40.5 kDa reference for the full-length alpha chain. |
| Signal peptide at residues 1–24 | Contributes to precursor mass before cleavage. |
| Signal-peptide cleavage | Produces a shorter mature alpha chain; its apparent mass is not supplied. |
| N-linked glycosylation at Asn110 | May raise apparent size; a visible shift is not established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | The membrane protein may be poorly recovered or solubilized. | Check membrane extraction and a known positive lysate. |
| Band higher than expected | Asn110 glycosylation may affect migration; the cause is unconfirmed. | Compare with a validated positive control and assess glycosylation if needed. |
| Band lower than expected | Signal-peptide cleavage may yield a smaller mature chain. | Confirm identity with an independent antibody or a positive control. |
| Multiple bands | Precursor processing or Asn110 glycosylation may contribute, but distinct bands are unverified. | Compare positive and negative controls before assigning bands. |
| Weak or no signal | Membrane-protein recovery or target abundance may be low. | Check extraction, loading and a known positive lysate. |
Experiment evidence
Western blot images showing HLA-B band
M00186-2 · HLA-B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HLA-B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HLA-B in Western blot, you can use appendix tissue, which shows high HPA expression.
Positive control: Appendix (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: HLA-B is membrane-bound, so efficient membrane-protein extraction may be needed for a clear signal.
HPA tissue expression evidence for HLA-B
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 |
|---|---|---|---|---|
| Appendix | endocrine cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial 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 → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | follicle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HLA-B Western Blot Tips
Deeper troubleshooting and optimisation questions for HLA-B, answered from its protein features.
How should HLA-B 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 annotated HLA-B isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands. Assess other possibilities without labeling bands as splice isoforms on this evidence.
How should Asn110 glycosylation be considered when interpreting bands?
PTM · UniProt lists one N-linked glycosylation site at Asn110, numbered in the full 362-residue sequence. Compare bands with that numbering in mind; an additional or slower band cannot be assigned to glycosylation from this feature alone.
Does this guide establish induction of HLA-B?
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-B?
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 M00186-2 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 HLA-B quantitation measure?
Quantitation · HLA-B is the alpha chain of a complex containing B2M and a peptide. Quantify the HLA-B alpha-chain signal consistently across samples; a Western blot band for that chain alone does not measure the amount of assembled peptide–HLA-B–B2M complex.
Why might HLA-B migrate differently from its predicted 40.5 kDa?
Interpretation · The 40.5 kDa prediction is for the 362-residue sequence. UniProt lists a signal peptide at residues 1–24 and one N-linked glycosylation site at Asn110. Processing and glycosylation could affect apparent mass, but these features alone do not establish a visible shift or explain a particular band.
UniProt lists two disulfide bonds. Consider whether samples were prepared under reducing or nonreducing conditions when comparing band patterns. The listed bonds do not predict a specific extra band or migration shift.
Start with the listed features: a 1–24 signal peptide, N-linked glycosylation at full-sequence Asn110, two disulfide bonds, and membrane localization. These suggest possibilities to investigate, but none identifies an unexpected band by itself. The supplied record gives no observed band size for comparison.
UniProt lists two disulfide bonds. Consider whether samples were prepared under reducing or nonreducing conditions when comparing band patterns. The listed bonds do not predict a specific extra band or migration shift.
Start with the listed features: a 1–24 signal peptide, N-linked glycosylation at full-sequence Asn110, two disulfide bonds, and membrane localization. These suggest possibilities to investigate, but none identifies an unexpected band by itself. The supplied record gives no observed band size for comparison.
Boster reagents
HLA-B 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 B7 Rabbit Monoclonal Antibody
Cat # M00186-2
M00186-2 is a human-reactive rabbit monoclonal antibody listed for HLA-B. Its Western blot image shows HLA B7 expression in Ramos cell lysate. No publication evidence or other tested specimens are supplied.
Which to pick: M00186-2 is the only listed option. Its Western blot image uses Ramos cell lysate; consider that tested context when choosing it for your samples.
Source: BosterBio HLA-B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P01889.
- Human Protein Atlas. HLA-B tissue expression.
- PMC13194846 — target-verified WB comparison
- PMC7339488 — target-verified WB comparison
- PMC3798713 — target-verified WB comparison