This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
BAD / Bcl2-associated agonist of cell death · Western blot design guide
Design a Western Blot for BAD
Real validated BAD Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-BAD 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
BAD 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 | ~18.4 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Bronchus (IHC candidate; verify WB) +4 more | |
| Negative control | Ovary (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated BAD Western Blot Protocols
The A03520 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | T24 cell lysates (catalog A03520) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A03520 · (A) 0.5, (B) 1, and (C) 2 μg/mL (catalog A03520) |
| 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 BAD Western Blot Band Size?
BAD has a predicted 18.4 kDa backbone; documented phosphorylation may produce nearby mobility shifts, but no empirical band size is supplied.
| Band near 18.4 kDa | Consistent with the predicted BAD backbone |
| Slightly slower migrating band | May reflect phosphorylation of BAD |
| Closely spaced doublet near 18.4 kDa | May reflect differently phosphorylated BAD forms |
| Several nearby bands | May reflect multiple phosphorylation states |
💡Expected BAD appearanceExpect BAD near its predicted 18.4 kDa, with possible nearby mobility shifts or a doublet reflecting its documented phosphorylation sites; no empirical band size is available.
| Predicted BAD mass | Places the unmodified backbone near 18.4 kDa |
| Ser25 phosphorylation | May alter apparent mobility |
| Ser75 phosphorylation | May alter apparent mobility |
| Ser91 phosphorylation | May alter apparent mobility |
| Ser97 phosphorylation | May alter apparent mobility |
| Ser99 phosphorylation | May alter apparent mobility |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | BAD may be enriched at the mitochondrial outer membrane | Check a mitochondrial fraction alongside whole-cell lysate |
| Band higher than expected | BAD phosphorylation may slow migration | Compare samples before and after phosphatase treatment |
| Band lower than expected | Possible BAD breakdown during preparation | Prepare fresh lysate with protease inhibitors |
| Multiple bands | Distinct BAD phosphorylation states may migrate differently | Compare bands before and after phosphatase treatment |
| Weak or no signal | BAD may partition between cytoplasm and mitochondrial outer membrane | Check both cytoplasmic and mitochondrial fractions |
| Fragments below expected size | BAD may have degraded during sample handling | Use fresh lysate and protease inhibitors |
Experiment evidence
Western blot images showing BAD band
A03520 · BAD
A03520-3 · BAD
M03520 · BAD
MP03520 · BAD
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for BAD Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for BAD in Western blot, you can use bronchus tissue lysate, which HPA scores as high.
Positive control: Bronchus (IHC candidate; verify WB)
Negative control: Ovary (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside BAD.
⚠️Feasibility: Ovary is an HPA not-detected tissue, though BAD may localize to either the mitochondrial outer membrane or cytoplasm.
HPA tissue expression evidence for BAD
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 |
|---|---|---|---|---|
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | cells in tubules | High | Protein (IHC) | HPA → |
| Nasopharynx | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced BAD Western Blot Tips
Deeper troubleshooting and optimisation questions for BAD, answered from its protein features.
Where should the BAD band appear?
Band shift · BAD has a predicted mass of 18.4 kDa. No observed Western blot band position is supplied, so use 18.4 kDa as a reference rather than an established apparent mass.
Should multiple BAD isoform bands be expected?
Isoforms · Only one isoform is listed. Multiple bands cannot be assigned to BAD isoforms from these features alone; assess them with an appropriate specificity control.
Could modifications shift BAD's apparent mass?
PTM · BAD has 12 listed modified residues and is annotated as phosphorylated, acetylated, and methylated. Compare treated and untreated samples if a band shifts; the supplied features do not establish a specific shift size or direction.
Phosphorylated BAD relocates to the cytoplasm, and the Ser-75/Ser-99 phosphorylated form binds 14-3-3 proteins. If comparing fractions or conditions, measure the same fraction across samples and distinguish total BAD from phosphorylation-specific signal.
Phosphorylated BAD relocates to the cytoplasm, and the Ser-75/Ser-99 phosphorylated form binds 14-3-3 proteins. If comparing fractions or conditions, measure the same fraction across samples and distinguish total BAD from phosphorylation-specific signal.
Does this guide establish induction of BAD?
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 BAD?
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 A03520 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 BAD be fractionated for quantitation?
Quantitation · BAD is annotated at the mitochondrial outer membrane and in the cytoplasm; phosphorylation is associated with cytoplasmic localization. Analyze comparable fractions across conditions, or include both fractions when assessing total BAD.
Do BAD's partners explain unexpected bands?
Interpretation · BAD forms heterodimers with Bcl-X(L), Bcl-2, and Bcl-W; its phosphorylated form binds 14-3-3 proteins. These interactions alone do not establish that an additional Western blot band is BAD. Verify unexpected bands with a specificity control.
Boster reagents
BAD 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-BAD Antibody
Cat # A03520
Real WB data
Anti-BAD Antibody Picoband®
Cat # A03520-3
Real WB data
Anti-Bad Rabbit Monoclonal Antibody
Cat # M03520
Real WB data
Anti-Phospho-Bad (S112) Monoclonal Antibody
Cat # MP03520
Four the supplier antibodies have supplied Western blot images for BAD. A03520 is shown in T24 lysates at three antibody concentrations; A03520-3 and M03520 have BAD blot images. MP03520 targets phospho-BAD (S112), with HeLa lysate examples. No publication or independent validation evidence is supplied.
Which to pick: For total BAD, consider A03520, A03520-3, or M03520; A03520 has the clearest supplied sample and concentration details. For phospho-BAD (S112), choose MP03520. Reactivity beyond the shown lysates is not specified.
Source: BosterBio BAD gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q92934.
- Human Protein Atlas. BAD tissue expression.
- PMC8992551 — target-verified WB comparison