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CD160 · Western blot design guide
Design a Western Blot for CD160
Real validated CD160 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CD160 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CD160 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 | ~19.8 kDa | |
| Observed band | ~27 kDa | |
| Gel | 12–15% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | N-linked glycosylation | |
| Regulation | Apical surface | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated CD160 Western Blot Protocols
Literature-validated Western blot parameters for CD160 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human PC-3 , Lane 3: human HEK293 , Lane 4: human A431 , Lane 5: human A549 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-CD160 antigen affinity purified polyclonal antibody (Catalog # A06168-1) 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 1:10000 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 CD160 at approximately 27KD. The expected band size for CD160 is at 20KD |
| Gel % | 12–15% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 27 kDa |
Section 2
What Is the Expected CD160 Western Blot Band Size?
CD160 has a 19.8 kDa predicted backbone but typically runs around 27 kDa on Western blots due to N-glycosylation at two sites, with possible higher bands from disulfide-linked homomultimers.
| single band around 27 kDa | the 19.8 kDa CD160 backbone plus mass added by N-linked glycosylation at Asn28 and Asn137 |
| diffuse or smeared band around 25-30 kDa | heterogeneous occupancy/processing of the two N-glycosylation sites |
| band near or below 19.8 kDa | mature protein after removal of the 1-24 signal peptide and the 160-181 propeptide during GPI-anchor attachment |
| higher-molecular-weight band at roughly double the monomer size | disulfide-linked homomultimer that did not fully dissociate under the reduction conditions used |
| extra band(s) at a different apparent mass than the main band | expression of one of the four annotated CD160 splice isoforms alongside the canonical form |
💡Expected CD160 appearanceExpect a CD160 band at about 27 kDa on reducing SDS-PAGE, higher than the 19.8 kDa predicted mass because of N-glycosylation at Asn28 and Asn137, with a fainter higher band possible if disulfide-linked homomultimers persist.
| predicted mass from UniProt (19.8 kDa, 181 aa) | sets the unmodified backbone size that glycosylation and other modifications shift the observed band away from |
| N-glycosylation at Asn28 and Asn137 | adds mass and heterogeneity, raising the apparent band to roughly 27 kDa and broadening it |
| signal peptide cleavage (residues 1-24) | removes N-terminal residues so the mature/processed protein runs smaller than the unprocessed full-length precursor |
| propeptide cleavage and GPI-anchor attachment (residues 160-181) | removes the C-terminal propeptide during lipid-anchor addition, altering the mature protein's apparent mass and its extraction/solubilization behavior |
| disulfide-linked homomultimer formation | produces a higher-order band at roughly twice the monomer mass when reduction is incomplete or samples are run non-reduced |
| alternative splice isoforms (1, 2, 3, 4) | can generate additional bands relative to the canonical isoform, differing qualitatively in size without a defined per-isoform mass |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | N-glycosylation at Asn28 and Asn137 adds mass beyond the 19.8 kDa predicted backbone | compare to a glycosidase-treated (e.g. PNGase F) sample to confirm the band shifts down toward the predicted mass |
| Broad smear instead of sharp band | heterogeneous glycan occupancy and processing at the two N-linked glycosylation sites | run a longer gradient gel and treat a parallel sample with glycosidase to sharpen the band |
| Multiple bands | co-expression of different CD160 splice isoforms and/or incompletely reduced disulfide-linked homomultimers | ensure full reduction with fresh reducing agent and compare band pattern against known isoform controls |
| Band lower than expected | removal of the signal peptide and propeptide during maturation and GPI-anchor attachment shrinks the protein relative to the unprocessed precursor | base size expectations on the processed mature protein rather than the full-length precursor mass |
| Fragments below expected size | proteolytic degradation of the disulfide-stabilized Ig domain or loss of the GPI-anchored region during sample handling | prepare lysates fresh with protease inhibitors and avoid repeated freeze-thaw cycles |
| Weak or no signal | GPI-anchored membrane protein may not be efficiently solubilized by mild lysis buffers | use a lysis buffer suited to membrane/GPI-anchored proteins and load adequate total protein |
Experiment evidence
Western blot images showing CD160 band
A06168-1 · CD160
A06168-1 · CD160
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CD160 Western blot
🧪For positive controls for CD160 in Western blot, you can use spleen tissue or purified NK cells/PBMCs, which are the classic sources of CD160-expressing lymphocytes.
Positive control: Spleen (or NK cells/PBMCs)
Negative control: Non-lymphoid cell line (e.g., HEK293); confirm with siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free imaging, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: CD160 is a GPI-anchored, cell-membrane protein restricted to NK cells and subsets of T lymphocytes, so whole-tissue lysates from non-lymphoid organs give little to no signal; enrich for lymphoid tissue or isolated NK/T cells, and since no HPA expression profile is available, verify specificity with siRNA knockdown or a CD160-KO line rather than relying on a presumed negative tissue.
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 CD160 Western Blot Tips
Deeper troubleshooting and optimisation questions for CD160, answered from its protein features.
Why does CD160 run at ~27 kDa, not 19.8 kDa?
The 19.8 kDa value is the unmodified core sequence. CD160 carries 2 N-glycosylation sites and is processed to a GPI-anchored mature protein after removal of the 1-24 signal peptide and 160-181 propeptide. These modifications add mass, producing the ~27 kDa apparent band commonly observed.
Will CD160 isoforms produce multiple bands?
UniProt lists 4 annotated isoforms from alternative splicing. Depending on which exons the antibody epitope falls within, one, several, or all isoforms may be detected, potentially as separate bands of differing size. Check the immunogen sequence against isoform-specific regions to predict which bands to expect.
Could N-glycosylation interfere with CD160 antibody binding?
With 2 predicted glycosylation sites near the extracellular Ig domain, glycans can mask nearby epitopes or cause lot-to-lot signal variability. If detection is weak or inconsistent, treat lysates with PNGase F; a resulting downward shift toward 19.8 kDa confirms glycosylation was affecting both mass and epitope access.
What blocking approach suits a glycosylated protein like CD160?
Standard 5% non-fat milk is generally adequate, but milk itself contains glycoproteins that can raise background with lectin-based or glycan-directed detection reagents. If using such reagents, switch to BSA blocking; for conventional antibody-based detection of CD160, milk blocking remains acceptable.
What transfer method to use for CD160 Western blot?
At its small ~20-27 kDa size, CD160 can blow through membranes during prolonged transfer. Use 0.2 micron PVDF rather than 0.45 micron, with a shortened semi-dry or wet transfer time (roughly 30-45 minutes), to retain the small glycosylated protein efficiently on the membrane.
What loading control fits CD160 quantitation?
CD160 is a cell membrane, GPI-anchor lipid-linked protein rather than a cytosolic one. When working with membrane-enriched fractions, normalize to a membrane-associated loading control rather than a cytosolic marker like GAPDH, since cytosolic controls do not reliably track membrane fraction loading or recovery.
What explains extra high-molecular-weight CD160 bands?
CD160 forms disulfide-linked homomultimers, stabilized by 2 disulfide bonds. Under non-reducing conditions this can produce dimer or multimer bands above the monomer size. Always run samples with a reducing agent such as DTT or beta-mercaptoethanol to resolve the true monomeric ~27 kDa species.
Could a lower molecular weight CD160 band appear?
CD160 keywords include both membrane and secreted forms. A soluble species lacking the GPI-anchor could migrate below the ~27 kDa membrane-bound band, particularly in serum, plasma, or conditioned media samples, and should not automatically be interpreted as nonspecific or degraded protein.
Boster reagents
Best CD160 Western Blot Antibodies
BosterBio's CD160 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-CD160 Antibody Picoband®
Cat # A06168-1
Our recommended anti-CD160 antibody is a best-performing, extensively cited reagent for Western blot, rigorously validated with orthogonal cross-validation against negative tissue controls and complementary detection methods, delivering dependable, reproducible results for confident CD160 protein detection.
Which to pick: Only one Boster CD160 antibody is listed, A06168-1, which includes a genuine Western blot validation image demonstrating specific detection, making it the clear, and only, choice for your CD160 Western blot experiments.
Source: BosterBio CD160 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95971.
- Human Protein Atlas. CD160 tissue expression.