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- Table of Contents
GSDME / Gasdermin-E · Western blot design guide
Design a Western Blot for GSDME
Real validated GSDME Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GSDME 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
GSDME 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 | ~54.6 kDa | |
| Observed band | ~55 kDa | |
| Gel | 8% (catalog A32407-2) | |
| 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 | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated GSDME Western Blot Protocols
The A32407-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human SH-SY5Y, human A549, human PC-3 (catalog A32407-2) |
| Gel % | 8% (catalog A32407-2) |
| Load | 30 ug; reducing conditions (catalog A32407-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A32407-2) |
| Membrane | nitrocellulose membrane (catalog A32407-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A32407-2) |
| Primary antibody | A32407-2 · 1:1000 (catalog A32407-2) |
| Primary incubation | overnight at 4°C (catalog A32407-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A32407-2) |
| Secondary incubation | 1.5 hour at RT (catalog A32407-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A32407-2) |
| Detection | ECL (catalog A32407-2) |
Section 2
What Is the Expected GSDME Western Blot Band Size?
GSDME is predicted at 54.6 kDa and observed at ~55 kDa; no cause for the small difference is established.
| Band at ~55 kDa | Matches the observed full-length GSDME band and its 54.6 kDa predicted mass |
| Additional bands at different sizes | Could reflect isoforms 1, 2, and 3; their migration is not specified |
| Slightly shifted band near ~55 kDa | Could reflect S-(2-succinyl)cysteine modification, though no visible shift is established |
| Higher-mass material | Could involve the annotated N-terminal membrane pore complex; its persistence on a blot is unestablished |
💡Expected GSDME appearanceFull-length GSDME is predicted at 54.6 kDa and observed at ~55 kDa in reducing whole-cell lysates; confirm band identity with a positive control and antibody specificity checks.
| Predicted full-length mass | 54.6 kDa, consistent with the observed ~55 kDa band |
| Isoforms 1, 2, and 3 | May differ in size, but their individual masses and migration are unspecified |
| S-(2-succinyl)cysteine at Cys45 | May affect apparent size; a visible shift is not established |
| S-(2-succinyl)cysteine at Cys156 | May affect apparent size; a visible shift is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Modification or retained N-terminal oligomer is possible, but neither is established as the cause | Compare reducing conditions and confirm identity with an independent antibody |
| Band lower than expected | A smaller GSDME species is possible; its identity is unspecified | Compare antibodies against different regions of GSDME |
| Multiple bands | Isoforms 1, 2, and 3 are annotated, but distinct bands are not established | Check band identity with isoform-aware reagents or knockdown |
| Weak or no signal | GSDME may be below detection in the tested sample | Include a GSDME-positive lysate and check sample loading |
| Fragments below expected size | A GSDME N-terminal species is annotated, without a supplied fragment mass | Compare N-terminal and C-terminal antibody signals |
Experiment evidence
Western blot images showing GSDME band
A32407-2 · GSDME
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GSDME Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for GSDME in Western blot, you can use breast tissue, where HPA reports 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 such as Ponceau alongside.
⚠️Feasibility: HPA lists no tissue as not detected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for GSDME
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 | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Prostate | glandular cells | Low | Protein (IHC) | HPA → |
| Urinary bladder | urothelial cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced GSDME Western Blot Tips
Deeper troubleshooting and optimisation questions for GSDME, answered from its protein features.
How should GSDME 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 might isoform 2 affect band interpretation?
Isoforms · UniProt lists residues 1–395 as missing in isoform 2, leaving residues 396–496 of the canonical sequence. An antibody targeting the missing region would not detect this isoform. Check the antibody epitope before assigning a smaller band.
UniProt lists residues 1–164 as missing in isoform 3. Its different sequence could produce a band distinct from full-length GSDME. Check whether the antibody epitope is retained before assigning that band to isoform 3.
UniProt lists residues 1–164 as missing in isoform 3. Its different sequence could produce a band distinct from full-length GSDME. Check whether the antibody epitope is retained before assigning that band to isoform 3.
Which listed modifications matter when assessing GSDME bands?
PTM · UniProt lists S-(2-succinyl)cysteine at positions 45, 156, 168, 180, 235, 371, 408, 417, and 489. These are canonical UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
Does this guide establish induction of GSDME?
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 GSDME?
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 A32407-2 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.
What should be quantified when comparing GSDME Western blots?
Quantitation · Quantify a consistently assigned band across samples. The ~55 kDa band is close to the canonical predicted mass, while isoforms 2 and 3 have missing N-terminal sequence. Keep distinct bands separate unless their identities are established; a combined signal could mix different GSDME species.
Why is the observed GSDME band near 55 kDa?
Interpretation · The canonical sequence has a predicted mass of 54.6 kDa, close to the reported ~55 kDa band. That agreement supports a full-length assignment but does not establish the band's identity by itself.
Consider the listed isoforms and the separately annotated GSDME N-terminal species. Compare band position and antibody epitope with the sequence retained in each isoform. The supplied features do not give a cleavage site, so they cannot identify a lower band as a specific fragment.
UniProt places the GSDME N-terminal species at the cell membrane and describes a membrane-inserted homooligomeric pore. When comparing samples or fractions, account for where that species is recovered; its signal need not track the full-length ~55 kDa band.
Consider the listed isoforms and the separately annotated GSDME N-terminal species. Compare band position and antibody epitope with the sequence retained in each isoform. The supplied features do not give a cleavage site, so they cannot identify a lower band as a specific fragment.
UniProt places the GSDME N-terminal species at the cell membrane and describes a membrane-inserted homooligomeric pore. When comparing samples or fractions, account for where that species is recovered; its signal need not track the full-length ~55 kDa band.
Boster reagents
GSDME 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-GSDME Antibody Picoband®
Cat # A32407-2
A32407-2 is a human-reactive anti-GSDME antibody with a Western blot image showing an approximately 55 kDa band in SH-SY5Y, A549, PC-3, and A431 whole-cell lysates. No publication or orthogonal validation evidence was supplied.
Which to pick: A32407-2 is the only listed option. Its WB image documents testing in four human cell lines using 30 µg lysate per lane and a 1:1000 primary antibody dilution; suitability for other samples is not established here.
Source: BosterBio GSDME gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O60443.
- Human Protein Atlas. GSDME tissue expression.
- PMC12649982 — target-verified WB comparison
- PMC9305212 — target-verified WB comparison
- PMC11965059 — target-verified WB comparison