Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (AB223877)

This Flow Cyt data was generated using the same anti-DFNA5/GSDME antibody clone [EPR19859] in a different buffer formulation (cat

ab215191).

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized SH-SY5Y (human neuroblastoma cell line from bone marrow) cell line labeling DFNA5/GSDMEwith ab215191 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (AB223877)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized EMT6 cell line labeling DFNA5/GSDME with ab215191 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

EMT6 cells were kindly provided by our collaborator Dr. Feng Shao, NIBS

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab215191).

• IP

Lab

Immunoprecipitation - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (AB223877)

ab215191 at 1/30 immunoprecipitating DFNA5/GSDME in EMT6 whole cell lysate.

Lane 1 (input) : EMT6 whole cell lysate (10μg)

Lane 2 (+) : ab215191 + EMT6 whole cell lysate

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab215191 in EMT6 whole cell lysate

For western blotting, ab215191 at 1/500 and ab131366 VeriBlot for IP Detection Reagent (HRP) was used at 1/1000.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

EMT6 cells were kindly provided by our collaborator Dr. Feng Shao, NIBS

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab215191).

All lanes:

Immunoprecipitation - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (<a href='/en-us/products/primary-antibodies/dfna5-gsdme-antibody-epr19859-n-terminal-ab215191'>ab215191</a>)

Predicted band size: 54 kDa

false

Exposure time: 55s

• WB

PubMed

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (AB223877)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab215191).

https://www.sciencedirect.com/journal/biochemical-and-biophysical-research-communications

All lanes:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (ab223877) at 1/1000 dilution

Lane 1:

Wild Type SGC-7901 cell lysates

Lane 2:

DFNA5/GSDME Knockout SGC-7901 cell lysates

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-ab6721'>ab6721</a>) at 1/2000 dilution

Predicted band size: 54 kDa

false

Image from Wang Y et al., Biochem Biophys Res Commu. 2018;495(1):1418-1425. Fig 4(A).; 10.1016/j.bbrc.2017.11.156 with permission from Elsevier.

• WB

Lab

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (AB223877)

This data was developed using ab215191, the same antibody clone in a different buffer formulation. Different batches of ab215191 were tested on SH-SY5Y (Human neuroblastoma epithelial cell) lysate at 0.6 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 55 kDa.

All lanes:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (<a href='/en-us/products/primary-antibodies/dfna5-gsdme-antibody-epr19859-n-terminal-ab215191'>ab215191</a>)

Predicted band size: 54 kDa

false

• WB

Lab

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - BSA and Azide free (AB223877)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : Lanes 1-3 : 10 seconds , Lanes 4-6 : 40 seconds.

Both ab215191 and ab221843 show negative result in MCF7, which is also consistent with the predicted expression pattern.

This data was developed using ab215191, the same antibody clone in a different buffer formulation.

Lanes 1 and 4:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (<a href='/en-us/products/primary-antibodies/dfna5-gsdme-antibody-epr19859-n-terminal-ab215191'>ab215191</a>) at 1/1000 dilution

Lanes 2 and 5:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859-60] - C-terminal (<a href='/en-us/products/primary-antibodies/dfna5-gsdme-antibody-epr19859-60-c-terminal-ab221843'>ab221843</a>) at 1/1000 dilution

Lanes 1 - 3:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lanes 4 - 6:

MCF7 (Human breast 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: 54 kDa

Observed band size: 55 kDa

false