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- Table of Contents
PAK4 / Serine/threonine-protein kinase PAK 4 · Western blot design guide
Design a Western Blot for PAK4
Real validated PAK4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PAK4 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
PAK4 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 | ~64.1 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Lung (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Methylated | |
| Caveat | PAK4/5/6 cross-reactivity | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated PAK4 Western Blot Protocols
The A01723-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | H1792 (20ug), MCF-7 (20ug), PC3 (20ug) (catalog A01723-1) |
| 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 | A01723-1; 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 PAK4 Western Blot Band Size?
PAK4 has a predicted mass of 64.1 kDa and four named isoforms; no empirical band or demonstrated isoform migration is supplied.
| Single band near 64.1 kDa | Consistent with the predicted mass of PAK4 isoform 1, pending identity controls |
| Several discrete bands | Could reflect PAK4 splice isoforms 1, 2, 3, and 4; their migration is not established |
| Doublet near the expected region | Could reflect different PAK4 forms, but the supplied features do not establish a resolvable doublet |
| Band away from 64.1 kDa | Could represent another PAK4 isoform; its mass and identity require confirmation |
💡Expected PAK4 appearanceUniProt predicts 64.1 kDa for PAK4, but no empirical band size is supplied; confirm any candidate band with a PAK4-specific identity control, especially with a PAK4/5/6 antibody.
| UniProt predicted mass | Places the reference sequence near 64.1 kDa; actual migration is unverified |
| Splice isoform 1 | May differ in size from other named isoforms; its individual migration is not supplied |
| Splice isoform 2 | May differ in size from other named isoforms; its individual migration is not supplied |
| Splice isoform 3 | May differ in size from other named isoforms; its individual migration is not supplied |
| Splice isoform 4 | May differ in size from other named isoforms; its individual migration is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | An alternative PAK4 isoform is possible, but its migration is unknown | Confirm band identity with PAK4 knockdown and an independent antibody |
| Band lower than expected | An alternative PAK4 isoform is possible, but its migration is unknown | Compare with PAK4 knockdown and an independent antibody |
| Multiple bands | Four PAK4 splice isoforms are annotated; distinct bands are unproven | Check which bands disappear after PAK4 knockdown |
| Broad smear instead of sharp band | PAK4 has annotated phosphorylation sites, but a smear from them is unproven | Compare phosphatase-treated and untreated samples and verify PAK4 identity |
| Weak or no signal | The candidate PAK4 signal may be below detection or antibody-dependent | Check loading, use a positive control, and verify antibody specificity |
Experiment evidence
Western blot images showing PAK4 band
A01723-1 · PAK4
A01723-1 · PAK4
MP01723 · PAK4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PAK4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PAK4 in Western blot, you can use adipose tissue lysate, which has medium HPA expression.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Lung (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: PAK4 is cytoplasmic, and HPA reports a usable tissue contrast between adipose tissue and lung.
HPA tissue expression evidence for PAK4
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lung | alveolar cells | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
| Appendix | glandular cells | Low | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Low | Protein (IHC) | HPA → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced PAK4 Western Blot Tips
Deeper troubleshooting and optimisation questions for PAK4, answered from its protein features.
How should PAK4 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 PAK4 isoforms produce different bands?
Isoforms · UniProt lists four isoforms. Relative to isoform 1, isoform 2 has E120K and lacks residues 121–285; isoform 3 lacks 69–221; isoform 4 lacks 132–221. These sequence differences could affect band position. The supplied features do not establish which isoforms are present in a sample.
Which PAK4 phosphorylation sites are listed?
PTM · In the supplied UniProt coordinates, phosphoserines occur at 41, 104, 148, 167, 181, 195, 258, 267, 291, and 474; phosphothreonines occur at 187 and 207. Ser474 is marked as phosphorylated by autocatalysis. Check an antibody’s numbering convention before matching its stated site to these coordinates.
Does this guide establish induction of PAK4?
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 PAK4?
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 A01723-1 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 multiple PAK4 bands be quantified?
Quantitation · The four listed isoforms include substantial sequence deletions. If bands are resolved, quantify each consistently and identify the isoform before combining signals. The supplied features give no antibody epitope or observed band positions, so band identity cannot be assigned from size alone.
Should PAK4 run at its predicted 64.1 kDa?
Interpretation · The supplied sequence predicts 64.1 kDa, but no observed band position is available. Use 64.1 kDa as a reference, not an expected apparent mass. The listed modifications alone do not establish a visible shift.
UniProt lists N6-methyllysine at position 78 in its coordinates. This identifies a modification to consider when checking an antibody epitope, but its presence alone does not demonstrate a detectable band shift.
UniProt places PAK4 in the cytoplasm and notes that it can appear at the cell periphery after activation of growth-factor or integrin-mediated signaling. If comparing fractions, keep the sampled compartment consistent; the supplied features do not establish a change in total PAK4 abundance.
Check the listed isoform deletions and the antibody’s target region when assessing additional bands. PAK4 also has phosphorylation and methylation sites, but their presence alone does not establish a visible shift or explain an observed mass difference. No empirical band position was supplied.
UniProt lists N6-methyllysine at position 78 in its coordinates. This identifies a modification to consider when checking an antibody epitope, but its presence alone does not demonstrate a detectable band shift.
UniProt places PAK4 in the cytoplasm and notes that it can appear at the cell periphery after activation of growth-factor or integrin-mediated signaling. If comparing fractions, keep the sampled compartment consistent; the supplied features do not establish a change in total PAK4 abundance.
Check the listed isoform deletions and the antibody’s target region when assessing additional bands. PAK4 also has phosphorylation and methylation sites, but their presence alone does not establish a visible shift or explain an observed mass difference. No empirical band position was supplied.
Boster reagents
PAK4 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-PAK4/5/6 (V469) Antibody
Cat # A01723-1
Real WB data
Anti-Phospho-PAK4 + PAK5 + PAK6 (S474 + S560 + S602) Monoclonal Antibody
Cat # MP01723
Two the supplier antibodies are listed for PAK4 Western blotting: A01723-1 targets PAK4/5/6, while MP01723 targets phosphorylated PAK4/5/6 sites. Both have WB images; the supplied evidence shows specific samples and does not establish PAK4-only detection.
Which to pick: Choose A01723-1 for PAK4/5/6 detection; its WB caption shows human cell lysates and mouse and rat testis lysates at 1:500. Choose MP01723 for the indicated phospho sites; its WB image shows U87-MG lysate only, despite listed human, mouse, and rat reactivity.
Source: BosterBio PAK4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O96013.
- Human Protein Atlas. PAK4 tissue expression.
- PMC5421896 — target-verified WB comparison
- PMC5620238 — target-verified WB comparison
- PMC3880571 — target-verified WB comparison