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CD58 · Western blot design guide
Design a Western Blot for CD58
Real validated CD58 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CD58 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CD58 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 | ~28.1 kDa | |
| Observed band | 35 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 | Heavy glycosylation | |
| Regulation | LPS-induced | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated CD58 Western Blot Protocols
Literature-validated Western blot parameters for CD58 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | glioma tissue vs adjacent non-cancerous tissue |
| Transfer | onto PVDF membrane (buffer/time/voltage not mentioned) |
| Membrane | PVDF (pore size not mentioned) |
| Blocking | BSA (buffer not mentioned) |
| Primary antibody | 1:1000 (CD58, Thermo PA5-78992) |
| Primary incubation | overnight, 4 °C |
Section 2
What Is the Expected CD58 Western Blot Band Size?
CD58 has a 28.1 kDa predicted core but runs at an observed ~35 kDa on Western blot due to extensive N-glycosylation at its six sites.
| prominent band at approximately 35 kDa | mature, fully N-glycosylated CD58 monomer running well above its 28.1 kDa unmodified core mass |
| diffuse or smeared band spanning roughly 30-40 kDa | heterogeneous occupancy and branching across the six N-linked glycosylation sites produces a mixture of glycoforms |
| band smaller than the calculated 28.1 kDa full-length translation product | cleavage of the 28-residue N-terminal signal peptide during membrane insertion removes several kDa from the precursor to yield the mature protein |
| two or more discrete bands at different apparent molecular weights | CD58 has three annotated splice isoforms of differing length, each capable of contributing a separate band |
| single clean band near 28 kDa | unglycosylated or fully deglycosylated CD58 core protein, seen after glycosidase treatment or in non-glycosylated expression systems |
💡Expected CD58 appearanceCD58 has a 28.1 kDa predicted core mass, but its six N-glycosylation sites shift the mature, membrane-bound protein to an observed native band around 35 kDa on reducing SDS-PAGE.
| Predicted core mass (28.1 kDa, 250 aa) | unmodified polypeptide baseline; rarely observed directly since the native protein is glycosylated |
| Six N-linked glycosylation sites (Asn40, Asn94, Asn109, Asn135, Asn169, Asn195) | cumulative glycan attachment raises the apparent mass to the observed ~35 kDa and can broaden the band |
| N-terminal signal peptide (residues 1-28) | cleaved upon membrane insertion, so the mature protein runs smaller than the full-length translated precursor |
| Alternative splicing (isoforms 1, 2, 3) | different isoforms vary in length, so lysates can show additional bands relatively larger or smaller than the canonical isoform |
| Non-glycosylated recombinant or bacterially expressed standard | lacking the six N-glycans, this form runs closer to the 28 kDa core mass, lower than native cell-derived CD58 |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | heavy N-glycosylation across six sites raises apparent mass well above the 28.1 kDa core | treat lysate with PNGase F and confirm the band collapses toward the core mass to verify glycan-dependent shift |
| Broad smear instead of sharp band | heterogeneous occupancy and branching of the six N-glycosylation sites generates a range of glycoforms | run a lower-percentage or longer gel for better resolution, or deglycosylate with PNGase F/Endo H to sharpen the band |
| Multiple bands | three alternatively spliced isoforms of differing length can be co-expressed | confirm which isoform is detected using isoform-specific epitope mapping and check isoform expression in the cell type used |
| Band lower than expected | signal peptide cleavage removes the 28-residue leader from the precursor, or the sample is a non-glycosylated recombinant construct | compare against the mature membrane-bound mass and verify the glycosylation state of the construct or standard used |
| No band in lysate | as a single-pass type I membrane glycoprotein, CD58 requires adequate detergent solubilization from the plasma membrane, and expression is low in some cell lines | use a membrane-compatible lysis buffer, confirm surface expression by flow cytometry, and include a known CD58-positive line such as HeLa as a positive control |
| Weak or no signal | low endogenous CD58 expression in certain cell types or antibody binding hindered by glycan masking of the epitope | use higher-expressing lines, increase protein loading, or select an antibody clone raised against a non-glycosylated region |
Experiment evidence
Western blot images showing CD58 band
RP1106 · CD58
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CD58 Western blot
🧪For positive controls for CD58 in Western blot, you can use human spleen tissue lysate or a lymphocyte-rich sample, reflecting CD58 (LFA-3) expression on immune cells, since no HPA expression data is available for this protein to pinpoint a specific tissue.
Positive control: Spleen (lymphoid tissue)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH, β-actin, and a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls alongside the target blot.
⚠️Feasibility: As a single-pass type I membrane protein with no HPA expression data to guide tissue selection, validate antibody specificity using a known CD58-expressing cell line and confirm signal loss with siRNA knockdown or a knockout line rather than relying on a tissue-based negative control.
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 CD58 Western Blot Tips
Deeper troubleshooting and optimisation questions for CD58, answered from its protein features.
Why does CD58 run heavier than its predicted mass?
CD58's predicted mass is 28.1 kDa for the mature polypeptide after signal peptide (residues 1-28) removal, but UniProt lists 6 N-glycosylation sites. Glycosylation on this Ig-domain membrane protein commonly adds several kDa of apparent mass, consistent with the observed ~35 kDa band. Confirm by treating a sample with PNGase F; deglycosylated CD58 should shift closer to 28 kDa.
Which CD58 isoform appears on a Western blot?
UniProt annotates 3 isoforms (1, 2, 3), but only isoform 1 is defined with the single-pass type I membrane topology and cell-membrane localization. Isoforms 2 and 3 may lack the transmembrane segment and run at a different apparent size. Verify your antibody's immunogen maps to isoform 1 sequence if a single ~35 kDa membrane-associated band is expected.
Does interferon-gamma signaling influence CD58 expression levels?
CD58/LFA-3 functions as the ligand for CD2 in immune cell adhesion, and antigen-presenting cell surface ligands in this pathway are commonly upregulated during interferon-gamma-driven immune activation. Expect increased band intensity, not a mass shift, in stimulated versus resting cell lysates. No UniProt-annotated modified residue accounts for this, so changes reflect expression level rather than post-translational modification.
What blocking buffer minimizes background for CD58 detection?
CD58 carries 6 glycosylation sites, so glycan-rich blocking reagents like nonfat milk can cause lectin-like nonspecific binding near the ~35 kDa glycoprotein band. Use a BSA-based blocking buffer instead to reduce background while preserving epitope accessibility on this heavily glycosylated single-pass membrane protein.
What transfer method to use for CD58 Western blot?
As a single-pass type I membrane glycoprotein, CD58 can transfer inefficiently with standard settings. Use wet transfer with reduced methanol (~10%) or semi-dry transfer with extended time to accommodate the glycosylated ~35 kDa species, since heavy glycosylation and membrane-embedded topology can slow migration out of the gel.
Which loading control best normalizes CD58 quantitation?
CD58 is a single-pass transmembrane protein localized to the cell membrane, so total-protein or membrane-fraction-specific loading controls give more accurate normalization than cytosolic housekeeping proteins in membrane-enriched preparations. If using whole-cell lysate, confirm consistent membrane protein recovery across samples before quantifying the ~35 kDa signal.
What explains extra bands near CD58's main band?
CD58 has one annotated disulfide bond, which can support dimerization or aggregation under non-reducing conditions, producing higher-molecular-weight bands. Always run samples with a reducing agent (DTT or beta-mercaptoethanol) to resolve the monomeric ~35 kDa band and distinguish it from disulfide-linked complexes or partial isoform-related species.
Boster reagents
Best CD58 Western Blot Antibodies
BosterBio's CD58 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-LFA3/CD58 Antibody Picoband®
Cat # RP1106
For CD58 (LFA-3) Western blotting, Boster's rabbit polyclonal RP1106 is the recommended choice, with a validated blot showing specific detection at 35 kDa in HeLa and K562 whole-cell lysates — clear, reproducible band-size confirmation for confident use.
Which to pick: Only one Boster CD58 antibody is catalogued: RP1106, a rabbit anti-LFA-3 affinity-purified polyclonal with an actual WB image (HeLa and K562 lysates, band at 35 kDa). Since it's the sole option listed, it's the default pick for WB.
Source: BosterBio CD58 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P19256.
- Human Protein Atlas. CD58 tissue expression.