This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
KL / Klotho · Western blot design guide
Design a Western Blot for KL
Real validated KL Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KL 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
KL 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 | ~116.2 kDa | |
| Gel | 8–10% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Isoform 1 shedding | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated KL Western Blot Protocols
The M02616-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human fetal kidney lysate (catalog M02616-1) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M02616-1; 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 KL Western Blot Band Size?
Klotho’s predicted full-length mass is 116.2 kDa; glycosylation, signal-peptide cleavage and homodimerization could affect migration, but no empirical band position is supplied.
| Band near 116.2 kDa | consistent with the predicted full-length precursor; confirm identity with antibody controls |
| Band above 116.2 kDa | may reflect N-linked glycosylation at annotated asparagine sites; a visible shift is not established |
| Band near twice the monomer size under non-reducing conditions | could reflect the annotated homodimer if it remains intact during electrophoresis |
| Band below 116.2 kDa | could reflect signal-peptide removal or another product; its size is not supplied |
| Several bands of different sizes | isoforms 1 and 2 are annotated, but their migration differences are not established |
| Weak or absent band in whole-cell lysate | a shed or secreted Klotho pool may be outside the lysate |
💡Expected KL appearanceThe 116.2 kDa value is the predicted full-length precursor mass; no empirical band size is supplied, so verify any candidate band with appropriate identity controls.
| Predicted full-length mass | 116.2 kDa is the sequence-based precursor reference, not a validated blot position |
| N-linked glycosylation at Asn 106, 159, 283, 344, 607, 612 and 694 | occupancy could raise or vary apparent mass; the size of any shift is unknown |
| Homodimer formation | an intact dimer could appear near twice the monomer mass under non-reducing conditions |
| Isoforms 1 and 2 | may differ in size, but their masses and migration difference are not supplied |
| Signal peptide at residues 1–33 | cleavage makes the mature chain smaller than the full-length precursor; its apparent mass is unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Klotho may be shed or secreted | check conditioned medium alongside lysate and include an identity control |
| Band higher than expected | N-linked glycosylation or an intact homodimer may contribute | compare deglycosylated samples and reducing conditions |
| Band lower than expected | signal-peptide cleavage or an unassigned product may contribute | compare bands with a validated Klotho control |
| Broad smear instead of sharp band | variable N-linked glycosylation is possible | compare untreated and deglycosylated samples |
| Multiple bands | isoforms, processing or differing glycosylation may contribute | use an independent Klotho antibody or other identity control |
| Weak or no signal | a shed or secreted pool may reduce lysate signal | check conditioned medium and a positive control |
Experiment evidence
Western blot images showing KL band
M02616-1 · KL
A02616 · KL
A02616 · KL
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KL Western blot
🧪For positive controls for KL in Western blot, you can use no HPA-supported tissue or cell line from the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA tissue controls cannot be selected from the supplied data, and KL shedding may shift some signal to extracellular samples.
HPA tissue expression evidence for KL
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced KL Western Blot Tips
Deeper troubleshooting and optimisation questions for KL, answered from its protein features.
How should KL 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.
How could the two KL isoforms differ on a blot?
Isoforms · Isoform 2 replaces residues 535..549 and lacks residues 550..1012 relative to isoform 1, so the isoforms have different sequences and lengths. Check whether the antibody’s target region is retained before assigning a band to either isoform.
Check the antibody’s stated epitope against the isoform sequences. Isoform 2 lacks UniProt residues 550..1012, so an epitope wholly within that region would not be present in isoform 2. The supplied features do not specify an antibody epitope.
Check the antibody’s stated epitope against the isoform sequences. Isoform 2 lacks UniProt residues 550..1012, so an epitope wholly within that region would not be present in isoform 2. The supplied features do not specify an antibody epitope.
Which KL glycosylation sites matter when interpreting bands?
PTM · UniProt annotates N-linked glycosylation at Asn106, Asn159, Asn283, Asn344, Asn607, Asn612, and Asn694. These are UniProt sequence coordinates; paper or antibody numbering may differ. Site annotations alone do not establish that a visible band shift will occur.
Does this guide establish induction of KL?
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 KL?
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 M02616-1 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 KL bands be quantified across samples?
Quantitation · Define which KL species the measurement covers and use the same antibody target and sample fraction across samples. KL has two sequence isoforms, and membrane isoform 1 can be shed. A signal from one fraction or epitope may therefore represent only part of the KL present.
Why might KL migrate differently from its predicted 116.2 kDa mass?
Interpretation · The 116.2 kDa prediction is for the sequence, while KL has a signal peptide at residues 1..33 and seven annotated N-linked glycosylation sites. These features can affect the protein present on a blot, but they do not establish an apparent band size or explain a specific discrepancy; no observed band size was supplied.
Isoform 1 is annotated as a single-pass cell membrane protein, and its shedding leads to a soluble form. Consider the membrane-containing sample and the soluble fraction when interpreting where a KL signal appears. The supplied features do not give the soluble form’s size.
Compare it with isoform structure, antibody epitope, sample fraction, and the seven annotated glycosylation sites. Isoform 2 lacks residues 550..1012, while shedding can yield soluble KL from membrane isoform 1. These features suggest possibilities but cannot identify a band without additional evidence.
Isoform 1 is annotated as a single-pass cell membrane protein, and its shedding leads to a soluble form. Consider the membrane-containing sample and the soluble fraction when interpreting where a KL signal appears. The supplied features do not give the soluble form’s size.
Compare it with isoform structure, antibody epitope, sample fraction, and the seven annotated glycosylation sites. Isoform 2 lacks residues 550..1012, while shedding can yield soluble KL from membrane isoform 1. These features suggest possibilities but cannot identify a band without additional evidence.
Boster reagents
KL 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-Klotho Rabbit Monoclonal Antibody
Cat # M02616-1
Real WB data
Anti-KLOTHO Antibody
Cat # A02616
Two the supplier anti-KL antibodies have WB images: M02616-1 with human fetal kidney lysate, and A02616 with human recombinant KLOTHO (30 ng per lane; band around 72 kDa). These images document those specific test contexts, not broader sample validation.
Which to pick: For human fetal kidney lysate, M02616-1 has the directly relevant WB image and lists human and mouse reactivity. A02616 lists human, mouse, and rat reactivity, but its WB image uses human recombinant protein; it is the listed option for rat samples.
Source: BosterBio KL gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UEF7.
- Human Protein Atlas. KL tissue expression.
- PMC4013593 — target-verified WB comparison
- PMC4938740 — target-verified WB comparison
- PMC5994118 — target-verified WB comparison