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- Table of Contents
CD8B / T-cell surface glycoprotein CD8 beta chain · Western blot design guide
Design a Western Blot for CD8B
Real validated CD8B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CD8B 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
CD8B 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 | ~23.7 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Thymus (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 | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 8 isoform(s) |
Section 1
Real Curated CD8B Western Blot Protocols
The A07259 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Jurkat, Raw264.7, H9C2 (catalog A07259) |
| 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 | A07259; 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 CD8B Western Blot Band Size?
CD8B has a predicted 23.7 kDa precursor; cleavage, glycosylation and CD8A association could alter migration, but no empirical band size is supplied.
| Band near 23.7 kDa | Consistent with the predicted CD8B precursor mass; confirm identity with controls |
| Band below 23.7 kDa | Could reflect removal of the 1–21 signal peptide |
| Band above 23.7 kDa | Could reflect N-linked glycosylation at Asn102; the shift is unquantified |
| Higher band under nonreducing conditions | Could contain the disulfide-linked CD8A/CD8B heterodimer |
| Several bands at different positions | Could reflect CD8B isoforms; distinct migration is unconfirmed |
💡Expected CD8B appearanceUniProt predicts a 23.7 kDa CD8B precursor, but no empirical band size is supplied; processing, glycosylation and association with CD8A may affect migration, so confirm band identity with appropriate controls.
| Predicted CD8B precursor mass | 23.7 kDa is the sequence-based reference, not a measured band |
| N-linked glycosylation at Asn102 | May increase apparent size; the amount is unknown |
| Signal peptide at residues 1–21 | Cleavage makes mature CD8B smaller than the precursor |
| Disulfide-linked CD8A/CD8B heterodimer | May produce a higher band when the complex remains linked |
| Isoforms 1, 2, 3, 4, 5, 6, 7 and 8 | Alternative sequences may differ in size; their masses and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CD8B expression may be low, or membrane protein recovery may be poor | Check a CD8B-positive control and membrane protein extraction |
| Band higher than expected | A CD8A/CD8B disulfide-linked complex may remain under incomplete reduction | Compare reducing and nonreducing lanes and confirm CD8B identity |
| Band lower than expected | Signal peptide cleavage may lower mass relative to the precursor | Check a positive control and verify the antibody-recognized band |
| Multiple bands | Alternative CD8B isoforms or differing modification states are possible | Compare a positive control and verify band identity with an independent antibody |
| Broad smear instead of sharp band | Incomplete solubilization of membrane CD8B or sample degradation is possible | Optimize membrane protein extraction and sample handling |
Experiment evidence
Western blot images showing CD8B band
A07259 · CD8B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CD8B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CD8B in Western blot, you can use thymus tissue, which HPA rates High for CD8B.
Positive control: Thymus (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: A clear positive and negative tissue are available, but efficient cell-surface expression of CD8B requires CD8A.
HPA tissue expression evidence for CD8B
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 |
|---|---|---|---|---|
| Thymus | cortical cells | High | Protein (IHC) | HPA → |
| Appendix | lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Medium | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD8B Western Blot Tips
Deeper troubleshooting and optimisation questions for CD8B, answered from its protein features.
Why might an unexpectedly high CD8B band appear?
Band shift · CD8B forms a disulfide-linked heterodimer with CD8A at the cell surface. A higher band under nonreducing conditions could therefore represent a linked complex. Compare reducing and nonreducing samples before assigning it; the supplied evidence gives no observed band size.
How can CD8B isoforms affect band interpretation?
Isoforms · Eight isoforms are listed. Isoforms 3 and 6 lack residues 166–195, while several isoforms replace sequence near the C terminus. Compare the isoform expressed in your sample with the antibody’s binding region before assigning a band by size.
Check whether the antibody’s binding region overlaps canonical residues 165–210. The supplied isoform variants delete or replace parts of this region, so an antibody against it may detect some isoforms differently. Confirm the relevant isoform and binding region before comparing signals.
Check whether the antibody’s binding region overlaps canonical residues 165–210. The supplied isoform variants delete or replace parts of this region, so an antibody against it may detect some isoforms differently. Confirm the relevant isoform and binding region before comparing signals.
Could glycosylation account for multiple CD8B bands?
PTM · Canonical CD8B has an annotated N-linked glycosylation site at Asn102. Glycosylation is a plausible contributor to band differences, but this annotation alone cannot establish that multiple observed bands are glycoforms.
UniProt annotates phosphotyrosine at canonical residue 209. This residue lies in a region replaced in several isoforms, so specify the sequence numbering and isoform when assessing a phospho-specific signal. The annotation alone does not establish a visible mobility shift.
UniProt annotates phosphotyrosine at canonical residue 209. This residue lies in a region replaced in several isoforms, so specify the sequence numbering and isoform when assessing a phospho-specific signal. The annotation alone does not establish a visible mobility shift.
Does this guide establish induction of CD8B?
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 CD8B?
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 A07259 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.
What matters when quantifying cell-surface CD8B?
Quantitation · Isoform 1 is a membrane protein, and efficient cell-surface expression requires CD8A. Define whether the measurement is total CD8B or cell-surface CD8B, and account for CD8A availability when interpreting differences in surface signal.
Why might CD8B migrate differently from its predicted 23.7 kDa?
Interpretation · The 23.7 kDa prediction includes the 1–21 signal peptide. CD8B also has an N-linked glycosylation site at Asn102. These features can affect the mature protein’s mass, but the supplied evidence gives no observed band size or demonstrated shift.
Boster reagents
CD8B 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-CD8B Antibody
Cat # A07259
The catalog reports one anti-CD8B antibody, A07259, with reported human, mouse, and rat reactivity. Its WB image uses Jurkat, Raw264.7, and H9C2 whole cell lysates at 1:500. The supplied evidence covers these named samples only.
Which to pick: A07259 is the only listed option. It has a WB image for the three named cell lysates; check whether its reported reactivity and tested samples fit your experiment.
Source: BosterBio CD8B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P10966.
- Human Protein Atlas. CD8B tissue expression.
- PMC3606432 — target-verified WB comparison