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Anti-GSDMD Antibody Picoband™ (monoclonal, 6D11)

Figure 1. Western blot analysis of GSDMD using anti-GSDMD antibody (M02842).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human THP-1 whole cell lysates,
Lane 2: human SW620 whole cell lysates,
Lane 3: human Jurkat whole cell lysates,
Lane 4: human U87 whole cell lysates,
Lane 5: human PC-3 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-GSDMD antigen affinity purified monoclonal antibody (Catalog # M02842) at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for GSDMD at approximately 53 kDa. The expected band size for GSDMD is at 53 kDa.
Figure 2. IHC analysis of GSDMD using anti-GSDMD antibody (M02842).
GSDMD was detected in a paraffin-embedded section of human cervical cancern tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-GSDMD Antibody (M02842) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 3. IHC analysis of GSDMD using anti-GSDMD antibody (M02842).
GSDMD was detected in a paraffin-embedded section of human cervical intraepithelial neoplasia tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-GSDMD Antibody (M02842) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 4. IHC analysis of GSDMD using anti-GSDMD antibody (M02842).
GSDMD was detected in a paraffin-embedded section of human esophageal squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml mouse anti-GSDMD Antibody (M02842) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Figure 5. IF analysis of GSDMD using anti-GSDMD antibody (M02842).
GSDMD was detected in an immunocytochemical section of PC-3 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL mouse anti-GSDMD Antibody (M02842) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 6. Flow Cytometry analysis of Jurkat cells using anti-GSDMD antibody (M02842).
Overlay histogram showing Jurkat cells stained with M02842 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-GSDMD Antibody (M02842, 1 µg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10 µg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

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Product Information
Application Applications Legend: WB=Western Blot IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin-embedded Sections) IHC-F=Immunohistochemistry (Frozen Sections) IF=Immunofluorescence FC=Flow Cytopmetry IC=Immunochemistry ICC=Immunocytochemistry E=ELISA IP=Immunoprecipitation DB=Dot Blot CHIP=Chromatin Immunoprecipitation FA=Fluorescence Assay IEM=Immuno electron microscopy EIA=Enzyme Immunoassay	WB, IHC, IF, ICC, FC
Primary Accession	P57764
Host	Mouse
Isotype	Mouse IgG2b
Reactivity	Human
Clonality	Monoclonal
Format	Lyophilized
Description	Anti-GSDMD Antibody Picoband™ (monoclonal, 6D11) . Tested in Flow Cytometry, IF, IHC, ICC, WB applications. This antibody reacts with Human.
Reconstitution	Adding 0.2 ml of distilled water will yield a concentration of 500 µg/ml.

Additional Information
Gene ID	79792
Other Names	Gasdermin-D, Gasdermin domain-containing protein 1, Gasdermin-D, N-terminal, GSDMD-NT, Gasdermin-D, 13 kDa, 13 kDa GSDMD, Gasdermin-D, p40, GSDMD {ECO:0000303\|PubMed:26375003, ECO:0000312\|HGNC:HGNC:25697}
Calculated MW	53 kDa
Application Details	Western blot, 0.25-0.5 µg/ml, Human Immunohistochemistry(Paraffin-embedded Section), 2-5 µg/ml, Human Immunocytochemistry/Immunofluorescence, 5 µg/ml, Human Flow Cytometry, 1-3 µg/1x10^6 cells, Human
Contents	Each vial contains 4 mg Trehalose, 0.9 mg NaCl and 0.2 mg Na2HPO4.
Clone Names	Clone: 6D11
Immunogen	E.coli-derived human GSDMD recombinant protein (Position: M1-H484).
Purification	Immunogen affinity purified.
Storage	At -20°C for one year from date of receipt. After reconstitution, at 4°C for one month. It can also be aliquotted and stored frozen at -20°C for six months. Avoid repeated freezing and thawing.

Protein Information
Name	GSDMD {ECO:0000303\|PubMed:26375003, ECO:0000312\|HGNC:HGNC:25697}
Function	[Gasdermin-D]: Precursor of a pore-forming protein that plays a key role in host defense against pathogen infection and danger signals (PubMed:26375003, PubMed:26375259, PubMed:27281216). This form constitutes the precursor of the pore-forming protein: upon cleavage, the released N-terminal moiety (Gasdermin-D, N-terminal) binds to membranes and forms pores, triggering pyroptosis (PubMed:26375003, PubMed:26375259, PubMed:27281216).
Cellular Location	[Gasdermin-D]: Cytoplasm, cytosol. Inflammasome {ECO:0000250\|UniProtKB:Q9D8T2}. Note=In response to a canonical inflammasome stimulus, such as nigericin, recruited to NLRP3 inflammasone with similar kinetics to that of uncleaved CASP1 precursor. {ECO:0000250\|UniProtKB:Q9D8T2} [Gasdermin-D, N-terminal]: Cytoplasm, cytosol. Note=(Microbial infection) Upon infection by M.tuberculosis, localization to cell membrane is prevented by M.tuberculosis phosphatase PtpB that catalyzes dephosphorylation of phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol 4- phosphate, thereby inhibiting the membrane targeting of Gasdermin-D, N- terminal and subsequent cytokine release and pyroptosis [Gasdermin-D, C-terminal]: Cytoplasm, cytosol {ECO:0000250\|UniProtKB:Q9D8T2}
Tissue Location	Expressed in the suprabasal cells of esophagus, as well as in the isthmus/neck, pit, and gland of the stomach, suggesting preferential expression in differentiating cells

Research Areas

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Application Protocols

Provided below are standard protocols that you may find useful for product applications.

Western Blot Protocol

Blocking Peptides Protocol

Dot Blot Protocol

Immunohistochemistry Protocol

Immunofluorescence Protocol

Immunoprecipitation Protocol

Flow Cytomety Protocol

Cell Culture Protocol

Background

Gasdermin D is a member of the gasdermin family. Members of this family appear to play a role in regulation of epithelial proliferation. Gasdermin D has been suggested to act as a tumor suppressor. Alternatively spliced transcript variants have been described.

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