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- Table of Contents
CD4 / T-cell surface glycoprotein CD4 · Western blot design guide
Design a Western Blot for CD4
Real validated CD4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CD4 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
CD4 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 | ~51.1 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 | N-linked glycosylation | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CD4 Western Blot Protocols
The M00344 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | THP-1 cell lysate (catalog M00344) |
| 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 | M00344; 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 CD4 Western Blot Band Size?
CD4 has a 51.1 kDa predicted precursor backbone; signal-peptide cleavage lowers polypeptide mass, while N-linked glycans raise apparent monomer size and disulfide-linked dimers may appear.
| Band above 51.1 kDa or a diffuse higher band | N-linked glycans at Asn296 and Asn325 increase apparent size |
| Band near twice the monomer position under nonreducing conditions | Disulfide-linked CD4 homodimer |
| Band slightly below the full-length precursor position | Cleavage of the 25-residue signal peptide |
| Band near 51.1 kDa | CD4 backbone with little glycan-dependent shift |
💡Expected CD4 appearanceExpect mature CD4 as a glycosylated band above its 51.1 kDa precursor backbone estimate, with possible broadening; nonreducing conditions can reveal a disulfide-linked homodimer near twice the monomer position.
| 51.1 kDa predicted precursor mass | Provides the unglycosylated full-length backbone reference |
| N-linked glycosylation at Asn296 | Can increase apparent monomer size and broaden the band |
| N-linked glycosylation at Asn325 | Can further increase apparent monomer size and broaden the band |
| Signal peptide at residues 1–25 | Cleavage makes the mature polypeptide smaller than the full-length precursor |
| Disulfide-linked cell-surface homodimer | Can produce a band near twice the monomer position without complete reduction |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CD4 is a cell-surface membrane protein | Check that the sample contains CD4-expressing cells and recover the membrane fraction |
| Band higher than expected | N-linked glycans or incompletely reduced disulfide-linked homodimers | Compare reducing and nonreducing lanes and assess glycosylation |
| Band lower than expected | Signal-peptide cleavage or reduced glycan content | Compare mature CD4 with the full-length precursor estimate and assess glycosylation |
| Broad smear instead of sharp band | Variable glycosylation at Asn296 and Asn325 | Assess glycan contribution with a deglycosylated comparison |
| Multiple bands | Different glycosylation states or residual disulfide-linked dimers | Compare reducing conditions and glycan-treated material |
| Weak or no signal | Low recovery of membrane-localized CD4 | Check membrane-protein extraction and CD4 expression in the sample |
Experiment evidence
Western blot images showing CD4 band
M00344 · CD4
M00344-2 · CD4
M00344-2 · CD4
M00344-2 · CD4
M00344-2 · CD4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CD4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CD4 in Western blot, you can use appendix tissue lysate.
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 alongside the samples.
⚠️Feasibility: CD4 is a membrane protein, so adequate membrane protein extraction matters for detecting it.
HPA tissue expression evidence for CD4
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 | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Lymph node | non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | non-germinal center cells | Medium | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD4 Western Blot Tips
Deeper troubleshooting and optimisation questions for CD4, answered from its protein features.
How should CD4 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 multiple CD4 bands represent different isoforms?
Isoforms · Only one CD4 isoform is listed. Consider glycosylation or other annotated modifications before assigning multiple bands to isoforms. The supplied features alone cannot identify the cause of any particular band.
How might CD4 modifications affect band patterns?
PTM · CD4 has two glycosylation sites, three annotated modified residues, and phosphoprotein and palmitate keywords. These features can contribute to band differences, but the supplied record does not specify modification states or their expected shifts.
Does this guide establish induction of CD4?
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.
What transfer method to use for CD4 Western blot?
Transfer · CD4 is a single-pass membrane protein with a predicted mass of 51.1 kDa. Check that your transfer conditions recover proteins near this mass, and assess transfer with a total-protein stain. The supplied features do not establish one preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00344 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 CD4 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might CD4 migrate above its predicted 51.1 kDa?
Interpretation · CD4 has two annotated glycosylation sites, which can increase apparent molecular weight. Its 1–25 signal peptide is removed during maturation, so 51.1 kDa is only a starting reference. No observed band size is supplied; interpret shifts cautiously.
CD4 is a cell-surface protein and localizes to lipid rafts. Keep sample preparation consistent across lanes when comparing CD4 abundance, because differences in membrane recovery could affect the measured signal.
CD4 forms disulfide-linked homodimers at the cell surface. A higher-mass band could therefore reflect a dimer; compare reducing and nonreducing samples to investigate. The supplied features do not establish the apparent size of that band.
CD4 has a 1–25 signal peptide, so maturation changes its mass. A lower-mass band cannot be assigned from this feature alone. Check its reproducibility and antibody specificity before interpreting it as a CD4 form.
CD4 is a cell-surface protein and localizes to lipid rafts. Keep sample preparation consistent across lanes when comparing CD4 abundance, because differences in membrane recovery could affect the measured signal.
CD4 forms disulfide-linked homodimers at the cell surface. A higher-mass band could therefore reflect a dimer; compare reducing and nonreducing samples to investigate. The supplied features do not establish the apparent size of that band.
CD4 has a 1–25 signal peptide, so maturation changes its mass. A lower-mass band cannot be assigned from this feature alone. Check its reproducibility and antibody specificity before interpreting it as a CD4 form.
Boster reagents
CD4 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-CD4 Rabbit Monoclonal Antibody
Cat # M00344
Real WB data
Anti-CD4 Monoclonal Antibody [9H5A8]
Cat # M00344-2
Two the supplier anti-CD4 antibodies have supplied Western blot images: M00344 with THP-1 cell lysate and M00344-2 with human recombinant CD4. The supplied evidence does not establish performance in other sample types.
Which to pick: For THP-1 cell lysate, consider M00344, which has a matching WB image. For human recombinant CD4, consider M00344-2, shown at three protein loads. Choose based on your sample type; neither image establishes performance for other samples.
Source: BosterBio CD4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P01730.
- Human Protein Atlas. CD4 tissue expression.
- PMC1524797 — target-verified WB comparison