This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
LEFTY2 / Left-right determination factor 2 · Western blot design guide
Design a Western Blot for LEFTY2
Source-linked LEFTY2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LEFTY2 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
LEFTY2 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 | ~40.9 kDa | |
| Gel | 12–15% (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 | LEFTY1 cross-reactivity | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked LEFTY2 Western Blot Protocol Options
The M06558-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Caco2 cell lysate (catalog M06558-2) |
| 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 | M06558-2; 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 LEFTY2 Western Blot Band Size?
LEFTY2 has a predicted 40.9 kDa precursor; cleavage, Asn158 glycosylation, and isoforms could affect migration, but no empirical band or shift is established.
| Band near 40.9 kDa | Candidate full-length LEFTY2 precursor at its predicted mass; identity requires confirmation |
| Band below 40.9 kDa | Could reflect signal-peptide or propeptide cleavage |
| Little or no band in whole-cell lysate | LEFTY2 is secreted and may be more abundant in conditioned medium |
| Several bands | Processing or isoforms 1 and 2 could contribute, but distinct migration is unproven |
| Band above 40.9 kDa | N-linked glycosylation at Asn158 could affect migration if the site is occupied |
💡Expected LEFTY2 appearanceLEFTY2 has a predicted 40.9 kDa precursor, but no empirical band size is supplied; cleavage and Asn158 glycosylation may affect migration, and the LEFTY1 plus LEFTY2 antibody requires identity controls.
| Predicted precursor mass | 40.9 kDa is the sequence-based reference, not a measured band |
| Signal peptide at residues 1–21 | Cleavage could yield a smaller product than the full-length precursor |
| Propeptide at residues 22–76 | Cleavage could further reduce the mature product's size |
| N-linked glycosylation site at Asn158 | Occupancy could alter apparent mass; the size of any shift is unknown |
| Isoforms 1 and 2 | Their apparent sizes could differ, but distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | LEFTY2 is secreted | Check conditioned medium and a known positive sample |
| Band higher than expected | Asn158 N-linked glycosylation could affect migration | Compare glycosidase-treated and untreated samples and confirm band identity |
| Band lower than expected | Signal-peptide or propeptide cleavage could produce a smaller form | Check whether the antibody recognizes the retained region and confirm identity |
| Multiple bands | Processing or isoforms could contribute; the antibody also recognizes LEFTY1 | Use a LEFTY2-specific depletion or other identity control |
| Weak or no signal | Secreted LEFTY2 may be scarce in whole-cell lysate | Test concentrated conditioned medium alongside a positive control |
Experiment evidence
Western blot images showing LEFTY2 band
M06558-2 · LEFTY2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for LEFTY2 Western blot
🧪For positive controls for LEFTY2 in Western blot, you can use an HPA-confirmed positive sample once identified; none is supplied in the 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 alongside the samples.
⚠️Feasibility: LEFTY2 is secreted, so conditioned medium may be more suitable than whole-cell lysate.
HPA tissue expression evidence for LEFTY2
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 LEFTY2 Western Blot Tips
Deeper troubleshooting and optimisation questions for LEFTY2, answered from its protein features.
How should LEFTY2 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 LEFTY2 isoforms affect the blot?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 94–127 relative to the canonical sequence. An antibody may detect them differently depending on its epitope; the annotation alone does not predict their apparent band positions.
How should LEFTY2 glycosylation be interpreted?
PTM · UniProt annotates one N-linked glycosylation site at Asn158 in the canonical sequence. Consider this site when comparing bands, but do not assign a band shift to glycosylation from the annotation alone. Antibody or paper numbering may use a different sequence convention.
Does this guide establish induction of LEFTY2?
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 LEFTY2?
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 M06558-2 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.
Where should LEFTY2 be sampled for quantitation?
Quantitation · UniProt identifies LEFTY2 as secreted. Include an appropriate extracellular sample when measuring its abundance, and compare the same sample compartment across conditions. State which compartment was measured when reporting the result.
Why might LEFTY2 migrate differently from its predicted 40.9 kDa?
Interpretation · The 40.9 kDa prediction refers to the full sequence. UniProt annotates a signal peptide at residues 1–21, a propeptide at 22–76, and N-linked glycosylation at Asn158. These features could affect the mass of a detected form, but they do not establish a visible shift or explain any particular band.
UniProt lists four disulfide bonds. Record whether samples were run under reducing or nonreducing conditions when comparing bands. The feature count alone does not predict a specific migration pattern.
Check whether the detected region includes the signal peptide (1–21), propeptide (22–76), isoform 2 deletion (94–127), or canonical Asn158 glycosylation site. These annotations suggest possible forms to examine; none identifies an unexpected band without further evidence.
UniProt lists four disulfide bonds. Record whether samples were run under reducing or nonreducing conditions when comparing bands. The feature count alone does not predict a specific migration pattern.
Check whether the detected region includes the signal peptide (1–21), propeptide (22–76), isoform 2 deletion (94–127), or canonical Asn158 glycosylation site. These annotations suggest possible forms to examine; none identifies an unexpected band without further evidence.
Boster reagents
LEFTY2 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-LEFTY1 + LEFTY2 Rabbit Monoclonal Antibody
Cat # M06558-2
The catalog reports a rabbit monoclonal antibody against LEFTY1 and LEFTY2, with stated human, mouse, and rat reactivity. Its Western blot image uses Caco2 cell lysate; the supplied evidence does not show whether it distinguishes LEFTY2 from LEFTY1.
Which to pick: M06558-2 is the only listed option and has a Western blot image from Caco2 lysate. Check that its stated reactivity fits your sample, and consider its combined LEFTY1/LEFTY2 target when interpreting bands.
Source: BosterBio LEFTY2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O00292.
- Human Protein Atlas. LEFTY2 tissue expression.
- PMC11990000 — target-verified WB comparison
- PMC6089757 — target-verified WB comparison
- PMC12254735 — target-verified WB comparison