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- Table of Contents
CD53 / Leukocyte surface antigen CD53 · Western blot design guide
Design a Western Blot for CD53
Source-linked CD53 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CD53 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
CD53 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 | ~24.3 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Duodenum (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked CD53 Western Blot Protocol Options
The M06249 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Jurkat cell lysate (catalog M06249) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M06249; 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 CD53 Western Blot Band Size?
CD53 is predicted at 24.3 kDa; its two N-linked glycosylation sites could affect migration, but no empirical band position or visible shift is established.
| Band near 24.3 kDa | Could correspond to CD53 near its predicted sequence mass; identity requires confirmation. |
| Band above 24.3 kDa | N-linked glycosylation at Asn129 or Asn148 could increase apparent size. |
| Bands at different positions | Variable glycosylation is possible, but distinct CD53 bands are not established. |
| Broad smear | Variable glycosylation is possible, but a smear is not established by the listed sites. |
💡Expected CD53 appearanceCD53 has a predicted sequence mass of 24.3 kDa and N-linked glycosylation sites at Asn129 and Asn148; no empirical band position is supplied, so confirm any candidate band with identity controls.
| Predicted sequence mass | Provides a 24.3 kDa reference, not a validated migration position. |
| N-linked glycosylation at Asn129 | Could increase apparent size if the site is occupied. |
| N-linked glycosylation at Asn148 | Could increase apparent size if the site is occupied. |
| Glycosylation at both listed sites | Combined occupancy could affect migration; its magnitude is unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | N-linked glycosylation could alter migration. | Compare untreated and deglycosylated samples and confirm band identity. |
| Band lower than expected | The lower band's identity is unconfirmed. | Check with an independent CD53 antibody or CD53 depletion. |
| Broad smear instead of sharp band | Variable N-linked glycosylation is possible. | Compare untreated and deglycosylated samples. |
| Multiple bands | Variable glycosylation is possible; distinct CD53 forms are unconfirmed. | Test deglycosylation and verify bands by CD53 depletion. |
| Weak or no signal | Membrane-associated CD53 may be poorly recovered from the sample. | Check membrane extraction and include a CD53-positive control. |
Experiment evidence
Western blot images showing CD53 band
M06249 · CD53
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CD53 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CD53 in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Duodenum (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As a multi-pass membrane protein, CD53 may give a stronger signal in membrane-enriched lysate.
HPA tissue expression evidence for CD53
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 | lymphoid tissue | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Spleen | cells in white pulp | High | Protein (IHC) | HPA → |
| Tonsil | germinal center cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Duodenum | glandular cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD53 Western Blot Tips
Deeper troubleshooting and optimisation questions for CD53, answered from its protein features.
How should CD53 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 documented CD53 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It does not support assigning additional bands to documented CD53 isoforms.
Which CD53 modifications should I consider when interpreting a band?
PTM · UniProt lists N-linked glycosylation at asparagines 129 and 148, using UniProt sequence numbering. Compare untreated and deglycosylated samples to assess whether glycosylation contributes to the band pattern; the listed sites alone do not predict the result.
Does this guide establish induction of CD53?
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 CD53?
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 M06249 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.
How should I quantify CD53 across samples?
Quantitation · Use comparable membrane-protein recovery across samples and quantify the same defined CD53 band or band region consistently. The record provides a predicted mass of 24.3 kDa and two N-linked glycosylation sites, but no observed band size to prescribe a fixed quantitation window.
Should CD53 migrate at its predicted 24.3 kDa mass?
Interpretation · 24.3 kDa is the predicted mass, not a documented Western-blot position. CD53 has N-linked glycosylation sites at UniProt positions 129 and 148, but their presence alone does not establish a visible shift or explain any difference from 24.3 kDa. No observed band size was supplied.
CD53 is a multi-pass membrane protein located at the cell membrane and also reported at cell junctions and synapses. Include a sample preparation that retains membrane proteins when assessing its Western-blot signal.
First compare their pattern with the predicted 24.3 kDa mass and assess whether glycosylation at UniProt positions 129 and 148 contributes. The supplied record lists no alternative sequence and provides no observed band size, so band position alone cannot identify an unexpected band as CD53.
CD53 is a multi-pass membrane protein located at the cell membrane and also reported at cell junctions and synapses. Include a sample preparation that retains membrane proteins when assessing its Western-blot signal.
First compare their pattern with the predicted 24.3 kDa mass and assess whether glycosylation at UniProt positions 129 and 148 contributes. The supplied record lists no alternative sequence and provides no observed band size, so band position alone cannot identify an unexpected band as CD53.
Boster reagents
CD53 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-CD53 Monoclonal Antibody
Cat # M06249
The catalog reports one anti-CD53 monoclonal antibody, M06249, with reported human reactivity. Its Western blot image shows CD53 expression in Jurkat cell lysate; the supplied evidence does not establish performance in other samples.
Which to pick: M06249 is the only listed option. Choose it if your experiment matches its reported human reactivity; the supplied Western blot example uses Jurkat cell lysate.
Source: BosterBio CD53 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P19397.
- Human Protein Atlas. CD53 tissue expression.
- PMC11913036 — target-verified WB comparison
- PMC12963270 — target-verified WB comparison