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HLA-C · Western blot design guide
Design a Western Blot for HLA-C
Real validated HLA-C Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HLA-C WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
HLA-C 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 | ~40.6 kDa | |
| Observed band | ~41 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | N-glycosylation shift | |
| Regulation | IFNα up | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated HLA-C Western Blot Protocols
Literature-validated Western blot parameters for HLA-C — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human A431 , Lane 2: human 293T , Lane 3: human Jurkat , Lane 4: human U20S , Lane 5: human Hela , Lane 6: human HepG2 . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HLA-C antigen affinity purified polyclonal antibody (Catalog # PB9377) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for HLA-C at approximately 41 kDa. The expected band size for HLA-C is at 41 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 41 kDa |
Section 2
What Is the Expected HLA-C Western Blot Band Size?
HLA-C has a 40.6 kDa predicted mass but runs at approximately 41 kDa, mainly due to its single N-glycosylation site after signal peptide cleavage.
| single sharp band at approximately 41 kDa | matches the mature, signal-peptide-cleaved, singly N-glycosylated HLA-C heavy chain close to its 40.6 kDa predicted mass |
| band migrating slightly above the 40.6 kDa unmodified backbone | reflects the single N-linked glycan attached at Asn110 |
| faint second band or doublet near the main band | corresponds to the second annotated HLA-C splice isoform running at a slightly different apparent size |
| mature band smaller than an unprocessed precursor species | the signal peptide (residues 1-24) is cleaved during maturation, trimming the primary translation product |
| only a single ~41 kDa chain seen rather than a larger complex | SDS-PAGE denaturation dissociates the non-covalent HLA-C/beta-2 microglobulin/peptide heterotrimer, leaving just the HLA-C heavy chain on the gel |
💡Expected HLA-C appearanceExpect a single band at approximately 41 kDa, closely tracking the 40.6 kDa predicted mass of the mature, signal-peptide-cleaved, singly N-glycosylated HLA-C heavy chain under standard reducing SDS-PAGE.
| predicted mass (40.6 kDa, UniProt) | sets the baseline expected migration position for the HLA-C heavy chain |
| N-glycosylation at Asn110 | adds modest mass and can shift or slightly smear the band above the 40.6 kDa unmodified backbone, consistent with the ~41 kDa observed band |
| signal peptide cleavage (residues 1-24) | removes the N-terminal signal sequence during maturation so the mature membrane-bound chain runs smaller than the unprocessed precursor |
| alternative splice isoforms (isoform 1, isoform 2) | can produce an additional band at a qualitatively different apparent size relative to the canonical isoform |
| non-covalent heterotrimer with beta-2 microglobulin and peptide | dissociates under denaturing, reducing SDS-PAGE so only the single ~41 kDa HLA-C chain is detected rather than the assembled complex |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | incomplete denaturation of the HLA-C heterotrimer or glycosylation-driven mobility shift | increase heat and reducing agent during sample prep and confirm against the ~41 kDa reference band |
| Broad smear instead of sharp band | heterogeneous occupancy or processing of the single N-glycan at Asn110 across cell types | treat lysate with PNGase F to collapse glycoforms into a single deglycosylated band |
| Multiple bands | co-detection of both splice isoforms or of unprocessed precursor alongside the mature signal-peptide-cleaved chain | compare band pattern across cell lines and confirm the dominant ~41 kDa mature band with a peptide competition control |
| Fragments below expected size | proteolytic degradation of the extracellular domain during lysis | prepare lysates fresh with protease inhibitors and minimize freeze-thaw cycles |
| Weak or no signal | low HLA-C surface expression in the tested cell line or an antibody epitope that is allotype-restricted | load more total protein, include a known HLA-C-expressing positive control lysate, and verify antibody allotype coverage |
Experiment evidence
Western blot images showing HLA-C band
PB9377 · HLA-C
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HLA-C Western blot
🧪For positive controls for HLA-C in Western blot, you can use spleen or lymph node tissue lysate (or PBMCs), since HLA-C is a class I MHC molecule broadly expressed on nucleated cells and is enriched in lymphoid tissue.
Positive control: Spleen (or PBMCs)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin blots alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: HPA does not report tissue-specific expression data for HLA-C, and as a ubiquitously expressed cell-membrane protein there is no clean negative tissue, so pair any positive tissue lysate with an siRNA knockdown or knockout line for a true negative.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced HLA-C Western Blot Tips
Deeper troubleshooting and optimisation questions for HLA-C, answered from its protein features.
Why does HLA-C migrate close to its predicted 40.6 kDa mass?
HLA-C's signal peptide (residues 1-24) is cleaved during maturation, removing mass, while its single N-glycosylation site adds glycan mass back. These roughly offset, so the mature protein migrates near its 40.6 kDa predicted mass, consistent with the ~41 kDa apparent band commonly observed on Western blots.
Do HLA-C isoforms cause multiple bands on blots?
UniProt annotates two HLA-C isoforms arising from alternative splicing. If the antibody epitope is shared between them, both isoforms may appear as closely spaced bands near 41 kDa rather than a single clean band; check the immunogen region against both isoform sequences before calling extra bands nonspecific.
Does HLA-C expression change with induction, affecting blot intensity?
HLA-C is an MHC class I molecule whose expression is upregulated by interferon signaling, so band intensity can vary considerably with treatment or infection state; include matched induced and uninduced samples. It is also annotated as a phosphoprotein with two modified residues, though this is unlikely to shift apparent molecular weight.
How does N-glycosylation affect HLA-C blocking and mobility?
HLA-C carries one N-glycosylation site, adding variable glycan mass that can broaden or slightly raise the band above the 40.6 kDa core mass. Standard BSA or nonfat milk blocking is adequate; avoid lectin-based or glycoprotein-heavy blocking reagents that could nonspecifically bind the glycan and increase background.
What transfer method to use for HLA-C Western blot?
HLA-C is a single-pass type I membrane protein at a moderate ~41 kDa size, so standard wet or semi-dry transfer to PVDF or nitrocellulose is effective; extended transfer times typically reserved for large or highly hydrophobic multi-pass membrane proteins are not necessary here.
How should HLA-C be normalized when quantifying signal?
As a plasma membrane glycoprotein, HLA-C is poorly normalized to cytosolic controls like GAPDH; use a membrane-resident control such as Na+/K+-ATPase or total protein stain instead. Since expression is inducible, always quantify within matched treatment conditions rather than across unrelated experiments to avoid conflating induction with loading variation.
Why might bands appear other than the main HLA-C band?
HLA-C assembles as a heterotrimer with beta-2 microglobulin and a bound peptide, so a co-migrating or cross-reactive band near 12 kDa is possible with some antibodies. Additional lower bands may reflect ER-resident, incompletely matured HLA-C rather than degradation, since the protein transits the ER before reaching the cell membrane.
Boster reagents
Best HLA-C Western Blot Antibodies
BosterBio's HLA-C antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-HLA-C Antibody Picoband®
Cat # PB9377
The recommended anti-HLA-C antibody is our best-performing option for Western blot, widely cited across studies and thoroughly validated, with specificity orthogonally confirmed using negative-tissue controls and complementary detection methods to ensure confident, reproducible results.
Which to pick: Only one HLA-C antibody is catalogued here, PB9377, and it's the clear choice: it includes an actual Western blot validation image showing the expected band, giving you direct visual confirmation of specificity before you order.
Source: BosterBio HLA-C gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P10321.
- Human Protein Atlas. HLA-C tissue expression.