Anti-GSDMD antibody [EPR20859]

• WB

Supplier Data

Western blot - Anti-GSDMD antibody [EPR20859] (AB219800)

Exposure time : Lanes 1 and 2 : 3 minutes; Lanes 3 and 4 : 48 seconds; Lanes 5 and 6 : 3 minutes.

Blocking/Dilution buffer : 5% NFDM/TBST.

Expression level of GSDMD in whole normal brain lysate is low or undetectable (PMID : 32671214, PMID : 34975487), we recommend loading higher amount of lysate or using lower antibody dilution.

All lanes:

Western blot - Anti-GSDMD antibody [EPR20859] (ab219800) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 10 µg

Lane 5:

Rat liver lysate at 10 µg

Lane 6:

Rat brain lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

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• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-GSDMD antibody [EPR20859] (AB219800)

Flow cytometry overlay histogram showing Raw264.7 cells stained with ab219800 (red line). The cells were fixed with 80% Methanol and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab219800) (1x 10⁶ in 100μl at 0.2μg/ml (1/2)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GSDMD antibody [EPR20859] (AB219800)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling GSDMD with ab219800 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081] secondary antibody at 1/1000 dilution.

Confocal image showing cytoplasmic and nuclear staining in RAW 264.7 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Magenta).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] (AB219800)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse small intestine labelling GSDMD with ab219800 at a concentration of 0.1µ/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20mins.

ab219800 anti-GSDMD antibody [EPR20859] was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] (AB219800)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse small intestine labelling GSDMD with ab219800 at a concentration of 0.05µ/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab219800 anti-GSDMD antibody [EPR20859] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] (AB219800)

Immunohistochemical analysis of paraffin-embedded (A) Small intestine tissue from wild-type C57BL/6JGpt mice (B) Small intestine tissue from GSDMD knockout mice staining with ab219800 at 1/5000 dilution and ready-to-use Goat Anti-Rabbit IgG H&L (HRP) secondary. Counterstaining with hematoxylin. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins. Positive staining on (A) Small intestine tissue from wild-type C57BL/6JGpt mice and no staining on (B) Small intestine tissue from GSDMD knockout mice. The section was incubated with ab219800 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND™ RX instrument. The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and GSDMD-KO homozygous mice (Strain ID : T010437).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] (AB219800)

Immunohistochemical analysis of paraffin-embedded Wild type mouse small intestine (A) and GSDMD KO mouse small intestine (B) tissue labeling GSDMD with ab219800 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on epithelium of wild type mouse small intestine (A), no staining on GSDMD KO mouse small intestine (B) (PMID : 17350798) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

The tissue samples were kindly provided by our collaborator Dr. Feng Shao NIBS.

Heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 was performed before commencing with IHC staining protocol.

The tissue samples were kindly provided by Dr Feng Shao's lab, NIBS.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] (AB219800)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labelling GSDMD with ab219800 at 1/1000 (0.542 μg/ml) followed by ab214880 at a ready to use dilution. Low expression tissue : weak staining on mouse lung. The section was incubated with ab219800 at 4°C overnight. Counterstained with hematoxylin. Secondary antibody only control : Secondary antibody is ab214880 at a ready to use dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

Supplier Data

Immunoprecipitation - Anti-GSDMD antibody [EPR20859] (AB219800)

GSDMD was immunoprecipitated from 0.35 mg of RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate with ab219800 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab219800 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : RAW 264.7 whole cell lysate 10 μg (Input).

Lane 2 : ab219800 IP in RAW 264.7 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab219800 in RAW 264.7 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-GSDMD antibody [EPR20859] (ab219800)

Predicted band size: 53 kDa

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• WB

Lab

Western blot - Anti-GSDMD antibody [EPR20859] (AB219800)

ab215203 mainly recognizes N-terminal GSDMD and ab219800 mainly recognizes full length GSDMD.

Exposure time : Lanes 1-4 : 100 seconds, Lanes 5-6 : 40 seconds.

Lanes 1 - 4:

Western blot - Anti-cleaved N-terminal GSDMD antibody [EPR20829-408] (<a href='/en-us/products/primary-antibodies/cleaved-n-terminal-gsdmd-antibody-epr20829-408-ab215203'>ab215203</a>) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-GSDMD antibody [EPR20859] (ab219800) at 1/1000 dilution

Lanes 1, 3 and 5:

Untreated THP-1 (human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lanes 2, 4 and 6:

THP-1 (human epidermoid carcinoma cell line) treated with 50ng/ml TPA for 24 hours, then treated with 5ng/ml LPS for 3 hours and add 1&mu;g/ml BFA for another 3 h whole cell lysate at 20 µg

Secondary

Lanes 1, 2, 5 and 6:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Lanes 3 - 4:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/5000 dilution

Observed band size: 31 kDa

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• WB

Supplier Data

Western blot - Anti-GSDMD antibody [EPR20859] (AB219800)

In Western blot, Anti-GSDMD antibody [EPR20859] (ab219800) 1 : 1,000 dilution (0.582 ug/ml) and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) 1 : 1,000,000 dilution.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GSDMD antibody [EPR20859] (ab219800) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa,37 kDa

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Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-GSDMD antibody [EPR20859] (AB219800)

Blocking and diluting buffer : 5% NFDM /TBST

This antibody could detect full length, as well as a N-terminal fragment after stimulation.

The lysates were kindly provided by Dr Feng Shao's lab, NIBS.

All lanes:

Western blot - Anti-GSDMD antibody [EPR20859] (ab219800) at 1/2000 dilution

Lane 1:

iBMM (mouse immortalized bone marrow derived macrophages) treated with 500ng/ml Bsak plus 500ng/ml anthrax protective antigen (PA) for 2h. Cell lysate

Lane 2:

iBMM (mouse immortalized bone marrow derived macrophages) treated with 500ng/ml Bsak plus 500ng/ml anthrax protective antigen (PA) for 2h. Concentrated cell supernatant

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa,31 kDa

Observed band size: 31 kDa,53 kDa

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• WB

Supplier Data

Western blot - Anti-GSDMD antibody [EPR20859] (AB219800)

Exposure time : Lanes 1 and 2 : 15 seconds; Lanes 3 and 4 : 37 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

The antibody has strong reactivity with GSDMD with a few weak bands of undermined proteins.

The lysates were kindly provided by our collaborator Dr Feng Shao's lab, NIBS.

All lanes:

Western blot - Anti-GSDMD antibody [EPR20859] (ab219800) at 1/1000 dilution

Lane 1:

Wild-type mouse liver whole cell lysate at 20 µg

Lane 2:

GSDMD knockout mouse liver whole cell lysate at 20 µg

Lane 3:

Wild-type mouse lung whole cell lysate at 20 µg

Lane 4:

GSDMD knockout mouse lung whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

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