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BNIP3L / BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like · Western blot design guide
Design a Western Blot for BNIP3L
Real validated BNIP3L Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-BNIP3L 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
BNIP3L 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 | ~23.9 kDa | |
| Observed band | ~35 kDa | |
| Gel | 5–20% (catalog A03107-3) | |
| Positive control | Breast (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated BNIP3L Western Blot Protocols
The A03107-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human Jurkat, human K562 (catalog A03107-3) |
| Gel % | 5–20% (catalog A03107-3) |
| Load | 30 ug; reducing conditions (catalog A03107-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03107-3) |
| Membrane | nitrocellulose membrane (catalog A03107-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03107-3) |
| Primary antibody | A03107-3 · 0.5 μg/mL (catalog A03107-3) |
| Primary incubation | overnight at 4°C (catalog A03107-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03107-3) |
| Secondary incubation | 1.5 hour at RT (catalog A03107-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03107-3) |
| Detection | ECL (catalog A03107-3) |
Section 2
What Is the Expected BNIP3L Western Blot Band Size?
BNIP3L is predicted at 23.9 kDa, but an empirical band appears near 35 kDa; the cause of the difference is not established.
| Band near 35 kDa | Empirical BNIP3L band reported in reducing whole-cell lysates; identity warrants a knockdown control |
| Band near 23.9 kDa | Near the predicted sequence mass; identity requires confirmation |
| Additional bands at different positions | Could reflect isoforms 1 and 2, whose individual migration is unknown |
| Higher-mass band | Could reflect BNIP3L self-association if complexes persist during preparation |
| Little or no band in whole-cell lysate | Membrane-associated BNIP3L may be underrepresented after extraction |
💡Expected BNIP3L appearanceBNIP3L has a predicted sequence mass of 23.9 kDa, while antibody QC reports a band near 35 kDa in reducing lysates; the difference is unexplained, so confirm identity by knockdown.
| Predicted sequence mass | Sets a 23.9 kDa reference, although the reported band is near 35 kDa |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | May differ in size from isoform 1, but its individual mass and migration are not supplied |
| Self-association | Could yield higher-mass species if complexes persist; their migration is unverified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated BNIP3L may be poorly recovered | Check membrane extraction and probe a membrane-enriched fraction |
| Band higher than expected | The reported 35 kDa band exceeds the 23.9 kDa prediction for an unestablished reason | Confirm the band with BNIP3L knockdown and a second antibody |
| Band lower than expected | An isoform or an unrelated antibody target could produce the band; isoform migration is unknown | Check whether the band decreases after BNIP3L knockdown |
| Multiple bands | BNIP3L has two named isoforms and self-associates, but neither accounts for specific bands yet | Compare reducing samples and use BNIP3L knockdown to identify specific bands |
| Weak or no signal | Membrane-associated BNIP3L may be lost during lysate preparation | Check recovery in a membrane-enriched fraction and verify sample loading |
Experiment evidence
Western blot images showing BNIP3L band
A03107-3 · BNIP3L
M03107 · BNIP3L
A03107 · BNIP3L
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for BNIP3L Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for BNIP3L in Western blot, you can use breast tissue, which HPA rates as high expression.
Positive control: Breast (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA lists only low-expression negative candidates, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for BNIP3L
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 |
|---|---|---|---|---|
| Breast | myoepithelial cells | High | Protein (IHC) | HPA → |
| Colon | fibroblasts | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Low | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Low | Protein (IHC) | HPA → |
Section 3
Advanced BNIP3L Western Blot Tips
Deeper troubleshooting and optimisation questions for BNIP3L, answered from its protein features.
How should BNIP3L 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 BNIP3L isoforms affect band interpretation?
Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks residues 1–40 of isoform 1. Check whether the antibody epitope lies in that segment before interpreting a missing or additional band. The sequence difference predicts a shorter polypeptide but does not establish a distinct visible band.
Which BNIP3L phosphorylation sites matter when comparing bands?
PTM · UniProt lists phosphoserines at positions 62, 117, 118, 120 and 166 in the supplied canonical sequence. These are UniProt coordinates; antibody or paper numbering may differ. A band change alone cannot establish phosphorylation at any of these sites.
Does this guide establish induction of BNIP3L?
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 BNIP3L Western blot?
Transfer · BNIP3L is listed as a single-pass membrane protein at the mitochondrial outer membrane, endoplasmic reticulum and nuclear envelope. Check transfer efficiency across the expected 23.9 kDa mass and the approximately 35 kDa apparent band. The supplied features do not identify a uniquely suitable transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03107-3 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 BNIP3L be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might BNIP3L appear near 35 kDa instead of 23.9 kDa?
Interpretation · 23.9 kDa is the predicted mass, while approximately 35 kDa is an observed apparent band. The supplied features do not establish why they differ. Confirm band identity with a BNIP3L-specific control; do not attribute the difference to phosphorylation or membrane association from feature presence alone.
BNIP3L has several listed membrane locations. Use the same sample preparation and fraction across samples, and normalize within that preparation. If comparing a membrane fraction, verify fraction recovery before treating a band-intensity change as a change in total BNIP3L.
Check whether the antibody recognizes both isoforms, including isoform 2, which lacks residues 1–40. BNIP3L also self-associates, but that feature alone does not establish that a higher band is an oligomer. Compare appropriate identity controls and denaturing conditions before assigning an unexpected band; the supplied features list no glycosylation sites.
BNIP3L has several listed membrane locations. Use the same sample preparation and fraction across samples, and normalize within that preparation. If comparing a membrane fraction, verify fraction recovery before treating a band-intensity change as a change in total BNIP3L.
Check whether the antibody recognizes both isoforms, including isoform 2, which lacks residues 1–40. BNIP3L also self-associates, but that feature alone does not establish that a higher band is an oligomer. Compare appropriate identity controls and denaturing conditions before assigning an unexpected band; the supplied features list no glycosylation sites.
Boster reagents
BNIP3L 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-BNIP3L Antibody Picoband®
Cat # A03107-3
Real WB data
Anti-BNIP3L/Nix Rabbit Monoclonal Antibody
Cat # M03107
Real WB data
Anti-Bnip3L Antibody
Cat # A03107
Three the supplier anti-BNIP3L antibodies have WB images. A03107-3 shows human cell and rat and mouse kidney lysates, with a reported band near 35 kDa versus an expected 24 kDa. M03107 and A03107 show K562 lysate; A03107 includes peptide competition.
Which to pick: For mouse or rat samples, A03107-3 has the most directly relevant WB examples. For human K562 lysate, all three have images; choose by antibody format or the reported conditions. A03107 is listed for human reactivity only.
Source: BosterBio BNIP3L gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O60238.
- Human Protein Atlas. BNIP3L tissue expression.
- PMC11254729 — target-verified WB comparison
- PMC5432493 — target-verified WB comparison
- PMC13092853 — target-verified WB comparison