重组Anti-MLKL抗体[EPR17514]

• WB

Unknown

Western blot - Anti-MLKL antibody [EPR17514] (AB184718)

Lanes 1 - 4 : Merged signal (red and green). Green - ab184718 observed at 54 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab184718 was shown to react with MLKL in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255408 (knockout cell lysate ab263788) was used. Wild-type and MLKL knockout samples were subjected to SDS-PAGE. ab184718 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MLKL antibody [EPR17514] (ab184718) at 1/1000 dilution

Lane 1:

HUVEC cell lysate at 20 µg

Lane 2:

HT-29 cell lysate at 20 µg

Lane 2:

Western blot - Human MLKL knockout HeLa cell line (<a href='/products/cell-lines/human-mlkl-knockout-hela-cell-line-ab255408'>ab255408</a>)

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

MLKL knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 37 kDa,54 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] (AB184718)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling MLKL with ab184718 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Cytoplasmic staining on the lymphocytes of human tonsil is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MLKL antibody [EPR17514] (AB184718)

ab184718 staining MLKL in HT-29 (Human colorectal adenocarcinoma epithelial cell) cells by Immunocytochemistry (ICC). Cells were fixed with 100% Methanol. Samples were incubated with primary antibody at 1/200 dilution (6.5μg/ml). An AlexaFluor^® 488 Goat anti-Rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution (2μg/ml). ab195889 , Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used as the counterstain antibody (1/200 dilution, 2.5 μg/ml . DAPI was used as a nuclear counterstain. Confocal image showing cytoplasmic staining on HT-29 cell line.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] (AB184718)

Immunohistochemical analysis of paraffin-embedded Human colonic adenocarcinoma tissue labeling MLKL with ab184718 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Cytoplasmic staining on tumor cells of human colonic adenocarcinoma is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-MLKL antibody [EPR17514] (AB184718)

Lanes 1 - 4 : Merged signal (red and green). Green - ab184718 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab184718 was shown to recognize MLKL in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when MLKL knockout samples were examined. Wild-type and MLKL knockout samples were subjected to SDS-PAGE. ab184718 and ab8245 (loading control to GAPDH) were diluted to 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MLKL antibody [EPR17514] (ab184718) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

MLKL knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Huvec cell lysate at 20 µg

Predicted band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-MLKL antibody [EPR17514] (AB184718)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MLKL antibody [EPR17514] (ab184718) at 1/20000 dilution

Lane 1:

HUVEC (Human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Lane 2:

HT-29 (Human colorectal adenocarcinoma cells) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-MLKL antibody [EPR17514] (AB184718)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MLKL antibody [EPR17514] (ab184718) at 1/1000 dilution

All lanes:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 193 kDa,37 kDa,45 kDa,52 kDa,54 kDa,98 kDa

Observed band size: 115 kDa,193 kDa,35 kDa,37 kDa,45 kDa,52 kDa,54 kDa

false