Anti-GSDMD antibody [EPR20859] - BSA and Azide free

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-GSDMD antibody [EPR20859] - BSA and Azide free (AB239377)

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] - BSA and Azide free (AB239377)

This data was developed using ab219800, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] - BSA and Azide free (AB239377)

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). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab219800).

• WB

Lab

Western blot - Anti-GSDMD antibody [EPR20859] - BSA and Azide free (AB239377)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (<a href='/en-us/products/primary-antibodies/gsdmd-antibody-epr20859-ab219800'>ab219800</a>) 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] - BSA and Azide free (AB239377)

This data was developed using ab219800, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/gsdmd-antibody-epr20859-ab219800'>ab219800</a>) at 1/1000 dilution

Lane 1:

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

Lanes 2 - 3:

PC-3 (human prostate adenocarcinoma epithelial cell) 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: 37 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-GSDMD antibody [EPR20859] - BSA and Azide free (AB239377)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] (<a href='/en-us/products/primary-antibodies/gsdmd-antibody-epr20859-ab219800'>ab219800</a>) 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] - BSA and Azide free (AB239377)

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.

This data was developed using ab219800, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-GSDMD antibody [EPR20859] (<a href='/en-us/products/primary-antibodies/gsdmd-antibody-epr20859-ab219800'>ab219800</a>) 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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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GSDMD antibody [EPR20859] - BSA and Azide free (AB239377)

This data was developed using ab219800, the same antibody clone in a different buffer formulation. 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] - BSA and Azide free (AB239377)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab219800).

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

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

• IP

Supplier Data

Immunoprecipitation - Anti-GSDMD antibody [EPR20859] - BSA and Azide free (AB239377)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab219800).

All lanes:

Immunoprecipitation - Anti-GSDMD antibody [EPR20859] (<a href='/en-us/products/primary-antibodies/gsdmd-antibody-epr20859-ab219800'>ab219800</a>)

Predicted band size: 53 kDa

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