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KIF4A / Chromosome-associated kinesin KIF4A · Western blot design guide
Design a Western Blot for KIF4A
Source-linked KIF4A Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KIF4A 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
KIF4A 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 | ~139.9 kDa | |
| Observed band | ~150 kDa | |
| Gel | 8% (catalog M04486) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Oral mucosa (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked KIF4A Western Blot Protocol Options
The M04486 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U2OS, human PC-3 (catalog M04486) |
| Gel % | 8% (catalog M04486) |
| Load | 30 ug; reducing conditions (catalog M04486) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M04486) |
| Membrane | nitrocellulose membrane (catalog M04486) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M04486) |
| Primary antibody | M04486 · 1:500 (catalog M04486) |
| Primary incubation | overnight at 4°C (catalog M04486) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M04486) |
| Secondary incubation | 1.5 hour at RT (catalog M04486) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M04486) |
| Detection | ECL (catalog M04486) |
Section 2
What Is the Expected KIF4A Western Blot Band Size?
KIF4A is predicted at 139.9 kDa and observed near 150 kDa; the cause of the difference is not established.
| Band near 150 kDa | Empirical KIF4A band reported in reducing whole-cell lysates; confirm identity with a KIF4A control. |
| Band near 140 kDa | Near the 139.9 kDa predicted sequence mass; identity requires confirmation. |
| Two bands at different positions | Isoforms 1 and 2 are possible contributors, but their migration is unknown. |
| Single band without a doublet | Isoforms 1 and 2 need not resolve into separate bands. |
💡Expected KIF4A appearanceKIF4A has a predicted mass of 139.9 kDa and an empirical band near 150 kDa in reducing whole-cell lysates; the difference is unexplained, so confirm band identity with a KIF4A control.
| UniProt predicted mass | The sequence predicts 139.9 kDa; the reported apparent band is near 150 kDa. |
| Splice isoform 1 | Its apparent size relative to isoform 2 is not supplied. |
| Splice isoform 2 | Its apparent size relative to isoform 1 is not supplied. |
| Alternative splicing of isoforms 1 and 2 | Could produce different band positions, but distinct migration has not been established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | KIF4A may be poorly represented in the sampled whole-cell lysate. | Check a KIF4A-positive lysate and consider nuclear or cytoskeletal fractions. |
| Band higher than expected | The reported KIF4A band is near 150 kDa versus a 139.9 kDa predicted mass; the cause is unknown. | Compare with the reported band and confirm identity by KIF4A depletion. |
| Band lower than expected | An isoform could differ in size, but isoform migration is not supplied. | Confirm KIF4A identity by depletion and check antibody epitope coverage. |
| Broad smear instead of sharp band | KIF4A has annotated phosphorylation sites, but a smear is not established. | Compare phosphatase-treated and untreated samples and check transfer quality. |
| Multiple bands | Isoforms 1 and 2 may contribute, but distinct bands are not demonstrated. | Use KIF4A depletion to identify specific bands and check epitope coverage. |
| Weak or no signal | KIF4A localizes to nuclear and cytoskeletal compartments that may be underrepresented in the preparation. | Check a positive-control lysate and assess extraction of those compartments. |
Experiment evidence
Western blot images showing KIF4A band
M04486 · KIF4A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KIF4A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for KIF4A in Western blot, you can use appendix tissue, which HPA rates High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Oral mucosa (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA lists oral mucosa as not detected, but confirm its negative signal in the chosen lysate.
HPA tissue expression evidence for KIF4A
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 → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje 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 |
|---|---|---|---|---|
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced KIF4A Western Blot Tips
Deeper troubleshooting and optimisation questions for KIF4A, answered from its protein features.
What should be checked when unexpected KIF4A bands appear?
Band shift · Check antibody epitope coverage against both isoforms, especially the replaced 1086–1127 region and missing 1128–1232 region in isoform 2. Also consider the annotated phosphorylation sites. Neither isoform annotations nor modification sites alone identify an unexpected band; confirm its identity before assigning it to KIF4A.
How could KIF4A isoforms affect band interpretation?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residues 1086–1127 are replaced and residues 1128–1232 are missing, using canonical UniProt coordinates. An antibody recognizing the missing region would not be expected to detect isoform 2. The features do not establish where either isoform migrates on a blot.
Which KIF4A phosphorylation sites matter when assessing band shifts?
PTM · UniProt annotates phosphoserine at 394, 801, 810, 815, 951, 1001, 1013, 1017, 1028, 1126, 1186 and 1225, and phosphothreonine at 799, 995 and 1181. These are canonical UniProt coordinates; antibody or paper numbering may differ. Phosphorylation is a possibility to investigate, but the annotations do not show that it causes a visible shift.
Does this guide establish induction of KIF4A?
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 KIF4A Western blot?
Transfer · KIF4A is predicted at 139.9 kDa, with a supplied observed band near 150 kDa. Verify that the chosen transfer conditions move protein in that size range onto the membrane. The supplied UniProt features do not specify a transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M04486 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 KIF4A samples be compared quantitatively?
Quantitation · KIF4A is annotated in the nucleus matrix, cytoplasm, spindle, midbody and chromosomes. Compare like sample preparations and cellular fractions so differences in localization do not confound band intensity. Define whether the measurement represents total lysate or a particular fraction.
Why might KIF4A appear near 150 kDa instead of 139.9 kDa?
Interpretation · The supplied observed band is approximately 150 kDa, versus a predicted mass of 139.9 kDa. KIF4A has annotated phosphorylation sites and two isoforms, but those features alone do not establish the cause of the difference or demonstrate a visible shift.
Check whether the epitope overlaps canonical residues 1086–1127, which are replaced in isoform 2, or 1128–1232, which are missing in isoform 2. This determines whether the antibody sequence can recognize both listed isoforms. Use canonical UniProt coordinates when comparing epitope maps.
Check whether the epitope overlaps canonical residues 1086–1127, which are replaced in isoform 2, or 1128–1232, which are missing in isoform 2. This determines whether the antibody sequence can recognize both listed isoforms. Use canonical UniProt coordinates when comparing epitope maps.
Boster reagents
KIF4A 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-KIF4A Rabbit Monoclonal Antibody
Cat # M04486
The catalog reports one anti-KIF4A rabbit monoclonal antibody, M04486, with stated human and mouse reactivity. Its WB image shows a band near 150 kDa in human U2OS and PC-3 whole-cell lysates; the expected size is 140 kDa. Mouse WB evidence is not shown.
Which to pick: M04486 is the only listed option and has a WB image from human U2OS and PC-3 lysates. For mouse samples, reactivity is stated, but the supplied WB image does not test mouse material.
Source: BosterBio KIF4A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95239.
- Human Protein Atlas. KIF4A tissue expression.
- PMC10278490 — target-verified WB comparison
- PMC9809311 — target-verified WB comparison
- PMC9972622 — target-verified WB comparison