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- Table of Contents
KLF5 / Krueppel-like factor 5 · Western blot design guide
Design a Western Blot for KLF5
Real validated KLF5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KLF5 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
KLF5 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 | ~50.8 kDa | |
| Observed band | ~55 kDa | |
| Gel | 10% (catalog A00727-1) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Hippocampus (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated KLF5 Western Blot Protocols
The A00727-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human Hela, human CACO-2 (catalog A00727-1) |
| Gel % | 10% (catalog A00727-1) |
| Load | 30 ug; reducing conditions (catalog A00727-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00727-1) |
| Membrane | nitrocellulose membrane (catalog A00727-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00727-1) |
| Primary antibody | A00727-1 · 0.5 μg/mL (catalog A00727-1) |
| Primary incubation | overnight at 4°C (catalog A00727-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00727-1) |
| Secondary incubation | 1.5 hour at RT (catalog A00727-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00727-1) |
| Detection | ECL (catalog A00727-1) |
Section 2
What Is the Expected KLF5 Western Blot Band Size?
KLF5 is predicted at 50.8 kDa and observed near 55 kDa; the cause of the difference is not established.
| Band near 55 kDa | Matches the empirical KLF5 band; confirm identity with appropriate controls |
| Band near 51 kDa | Near the predicted 50.8 kDa mass; confirm identity with appropriate controls |
| Several bands near the expected region | Could reflect isoforms 1, 2, 3, or 4; their migration is unestablished |
| Weak band in whole-cell lysate | Nuclear KLF5 may be easier to detect after nuclear enrichment |
💡Expected KLF5 appearanceKLF5 has a predicted mass of 50.8 kDa and an empirical band near 55 kDa; the difference is unexplained, so confirm band identity with appropriate controls.
| Predicted KLF5 mass | 50.8 kDa provides a sequence-based reference, while the observed band is near 55 kDa |
| Isoform 1 | Its apparent size relative to the other named isoforms is unknown |
| Isoform 2 | May migrate differently from isoform 1; the direction is unknown |
| Isoform 3 | May migrate differently from isoform 1; the direction is unknown |
| Isoform 4 | May migrate differently from isoform 1; the direction is unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear KLF5 may be scarce in the sampled whole-cell lysate | Check a nuclear-enriched fraction and a positive control |
| Band higher than expected | The empirical KLF5 band is near 55 kDa rather than the predicted 50.8 kDa; the cause is unestablished | Compare with the reported 55 kDa band and confirm identity by KLF5 depletion |
| Band lower than expected | An alternative isoform is possible, but its mass is unknown | Confirm identity by KLF5 depletion and check antibody epitope coverage |
| Multiple bands | KLF5 has four named isoforms, but their migration patterns are unknown | Use KLF5 depletion and isoform-specific controls to assign bands |
| Weak or no signal | Nuclear localization may limit signal in whole-cell lysate | Enrich nuclear protein and check a positive control |
Experiment evidence
Western blot images showing KLF5 band
A00727-1 · KLF5
A00727 · KLF5
PA1889 · KLF5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KLF5 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for KLF5 in Western blot, you can use adipose tissue, which HPA scores High.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Hippocampus (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: KLF5 is nuclear, so nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for KLF5
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 | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Nasopharynx | respiratory epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Low | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Low | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Low | Protein (IHC) | HPA → |
Section 3
Advanced KLF5 Western Blot Tips
Deeper troubleshooting and optimisation questions for KLF5, answered from its protein features.
How should KLF5 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 KLF5 isoforms produce additional bands?
Isoforms · Four isoforms are listed. In UniProt canonical coordinates, isoform 2 lacks residues 88–239, isoform 4 lacks 1–91, and isoform 3 replaces 105–166 and lacks 167–457. These sequence differences could affect band size and antibody recognition; check whether the antibody epitope remains in each isoform.
Could modification affect KLF5 band interpretation?
PTM · The UniProt keywords include Ubl conjugation and isopeptide bond, but the supplied modified-residue list is empty. Do not assign a band shift or modification site from these features alone.
Does this guide establish induction of KLF5?
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 KLF5 Western blot?
Transfer · Choose transfer conditions appropriate for a protein near the predicted 50.8 kDa and observed ~55 kDa sizes. Check transfer at that region with total-protein staining before interpreting a weak or missing band.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00727-1 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 KLF5 bands be quantified?
Quantitation · Quantify a consistently identified band in the ~55 kDa region and normalize within the same sample preparation. Because four isoforms are listed, document which band or bands were measured and whether the antibody can recognize their sequences.
Does the ~55 kDa band match KLF5’s predicted mass?
Interpretation · KLF5’s canonical sequence has a predicted mass of 50.8 kDa, while the supplied observed band is about 55 kDa. The supplied features do not establish why they differ. Assess band identity using controls alongside apparent size.
KLF5 is annotated as nuclear. A nuclear fraction may improve detection when whole-cell lysate gives a weak signal. Check fraction quality before comparing signal between preparations.
Compare their sizes with the listed isoform sequence changes, and check whether the antibody epitope is retained. Nuclear localization and the ~55 kDa observed band offer additional context. An unexpected band cannot be assigned to an isoform or modification from size alone.
KLF5 is annotated as nuclear. A nuclear fraction may improve detection when whole-cell lysate gives a weak signal. Check fraction quality before comparing signal between preparations.
Compare their sizes with the listed isoform sequence changes, and check whether the antibody epitope is retained. Nuclear localization and the ~55 kDa observed band offer additional context. An unexpected band cannot be assigned to an isoform or modification from size alone.
Boster reagents
KLF5 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-KLF5 Antibody Picoband®
Cat # A00727-1
Real WB data
Anti-KLF5 Antibody (C-term)
Cat # A00727
Real WB data
Anti-Krueppel-like factor 5 KLF5 Antibody Picoband®
Cat # PA1889
Three the supplier anti-KLF5 antibodies have Western blot images from human cell lysates: A00727-1 in A549, HeLa, CACO-2 and PC-3; A00727 in CEM; and PA1889 in HeLa and A549. The supplied images do not establish performance in every listed species.
Which to pick: For mouse or rat samples, A00727-1 is the only listed antibody with stated reactivity, though its WB image uses human lysates. For human samples, choose by the closest tested lysate: A00727-1 covers four cell lines, A00727 CEM, and PA1889 HeLa and A549.
Source: BosterBio KLF5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q13887.
- Human Protein Atlas. KLF5 tissue expression.
- PMC11662046 — target-verified WB comparison
- PMC7555189 — target-verified WB comparison
- PMC3949508 — target-verified WB comparison