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- Table of Contents
KIF5B / Kinesin-1 heavy chain · Western blot design guide
Design a Western Blot for KIF5B
Real validated KIF5B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KIF5B 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
KIF5B 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 | ~109.7 kDa | |
| Observed band | ~110 kDa | |
| Gel | 10% (catalog A01934-2) | |
| 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 | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated KIF5B Western Blot Protocols
The A01934-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human HepG2, human Caco-2 (catalog A01934-2) |
| Gel % | 10% (catalog A01934-2) |
| Load | 30 ug; reducing conditions (catalog A01934-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01934-2) |
| Membrane | nitrocellulose membrane (catalog A01934-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01934-2) |
| Primary antibody | A01934-2 · 1:1000 (catalog A01934-2) |
| Primary incubation | overnight at 4°C (catalog A01934-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01934-2) |
| Secondary incubation | 1.5 hour at RT (catalog A01934-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01934-2) |
| Detection | ECL (catalog A01934-2) |
Section 2
What Is the Expected KIF5B Western Blot Band Size?
KIF5B is predicted at 109.7 kDa and observed at ~110 kDa; the small numerical difference reflects rounding, with no demonstrated migration shift.
| Band at ~110 kDa | Matches the observed KIF5B band and its 109.7 kDa predicted mass |
| Higher band under incomplete denaturation | May reflect retained assembly containing two KIF5B heavy chains; identity needs confirmation |
| Weak band in a soluble fraction | KIF5B can associate with the cytoskeleton and the cytoplasmic side of membranes |
| Doublet near ~110 kDa | Could involve the annotated Ser933 phosphorylation, but a visible shift is not established |
💡Expected KIF5B appearanceKIF5B is predicted at 109.7 kDa and observed at ~110 kDa in reducing whole-cell lysates; confirm band identity with appropriate antibody and KIF5B depletion controls.
| Predicted KIF5B mass | 109.7 kDa, consistent with the observed ~110 kDa band |
| N-acetylalanine at residue 2 | Adds a small modification; no visible migration effect is established |
| Phosphoserine at residue 933 | Adds a small modification; no visible migration effect is established |
| Omega-N-methylarginine at residue 956 | Adds a small modification; no visible migration effect is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoskeletal or membrane-associated KIF5B may be poorly recovered | Check extraction of cytoskeletal and membrane-associated material and include a positive lysate control |
| Band higher than expected | Incomplete denaturation may retain a heavy-chain assembly | Check denaturation conditions and verify the band with KIF5B depletion |
| Band lower than expected | The supplied features do not establish a smaller KIF5B product | Verify specificity with KIF5B depletion and check sample integrity |
| Multiple bands | A second band is not explained by a supplied isoform; Ser933 phosphorylation has no established visible shift | Compare bands after KIF5B depletion and assess phosphorylation only if a shift is reproducible |
| Weak or no signal | Recovery of cytoskeletal or membrane-associated KIF5B may be low | Check extraction and loading with a positive whole-cell lysate control |
Experiment evidence
Western blot images showing KIF5B band
A01934-2 · KIF5B
M01934 · KIF5B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KIF5B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for KIF5B in Western blot, you can use appendix tissue, which HPA rates High.
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: A tissue negative control is feasible because HPA reports KIF5B as not detected in adipose tissue.
HPA tissue expression evidence for KIF5B
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 | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | 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 → |
| Soft tissue | chondrocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced KIF5B Western Blot Tips
Deeper troubleshooting and optimisation questions for KIF5B, answered from its protein features.
How should KIF5B 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 a second KIF5B band be an annotated isoform?
Isoforms · The supplied features list one isoform and no alternative sequence. Do not assign a second band to an annotated KIF5B isoform on this evidence alone.
Which KIF5B modifications should I consider when interpreting bands?
PTM · UniProt lists N-acetylalanine at position 2, phosphoserine at 933, and omega-N-methylarginine at 956. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
Does this guide establish induction of KIF5B?
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 KIF5B Western blot?
Transfer · KIF5B has a predicted mass of 109.7 kDa. Check transfer of proteins near 110 kDa with a molecular-weight marker and a post-transfer gel or membrane stain, then adjust transfer conditions if that region transfers poorly. The supplied features do not specify a transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01934-2 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 the KIF5B band?
Quantitation · Quantify the band near 110 kDa within the detector's linear range and use a consistent loading reference across samples. KIF5B is associated with the cytoskeleton and is also reported at membranes, so keep sample preparation consistent when comparing its signal.
Why does KIF5B appear near 110 kDa?
Interpretation · The reported band near 110 kDa is close to the 109.7 kDa predicted mass. This agreement supports the assignment, but band position alone does not establish identity.
KIF5B is listed in the cytoplasmic cytoskeleton and at lysosome and cytolytic granule membranes. Its signal may depend on which cellular fraction is collected. Record whether each sample is a whole-cell lysate or a fraction when comparing bands.
KIF5B is described as part of an oligomer with two heavy and two light chains. An unexpected higher band should be checked for KIF5B identity and sample preparation effects; the oligomer annotation alone does not prove that the band contains an intact complex.
KIF5B is listed in the cytoplasmic cytoskeleton and at lysosome and cytolytic granule membranes. Its signal may depend on which cellular fraction is collected. Record whether each sample is a whole-cell lysate or a fraction when comparing bands.
KIF5B is described as part of an oligomer with two heavy and two light chains. An unexpected higher band should be checked for KIF5B identity and sample preparation effects; the oligomer annotation alone does not prove that the band contains an intact complex.
Boster reagents
KIF5B 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-KIF5B Antibody
Cat # A01934-2
Real WB data
Anti-KIF5B Rabbit Monoclonal Antibody
Cat # M01934
Two anti-KIF5B antibodies are listed for Western blotting, both with reported human, mouse, and rat reactivity and WB images. A01934-2 shows an approximately 110 kDa band in six named cell lysates; M01934 shows a HeLa lysate blot. No publication evidence is supplied.
Which to pick: Choose A01934-2 if you want documented WB conditions and examples from human, rat, and mouse cell lysates. Consider M01934 for a HeLa lysate example; its supplied caption gives no band size or protocol details.
Source: BosterBio KIF5B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P33176.
- Human Protein Atlas. KIF5B tissue expression.
- PMC7872075 — target-verified WB comparison
- PMC11279799 — target-verified WB comparison
- PMC6642081 — target-verified WB comparison