Anti-MLKL antibody [EPR17514] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MLKL antibody [EPR17514] - BSA and Azide free (AB211045)

Clone EPR17514 (ab211045) has been successfully conjugated by Abcam. This image was generated using Anti-MLKL antibody [EPR17514] (Alexa Fluor® 647). Please refer to ab207902 for protocol details.

ab207902 staining MLKL in SW480 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab207902 at a 1/50 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at a 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] - BSA and Azide free (AB211045)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184718).

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] - BSA and Azide free (AB211045)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184718).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MLKL antibody [EPR17514] - BSA and Azide free (AB211045)

Clone EPR17514 (ab211045) has been successfully conjugated by Abcam. This image was generated using Anti-MLKL antibody [EPR17514] (Alexa Fluor® 488). Please refer to ab207901 for protocol details.

ab207901 staining MLKL in SW480 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab207901 at a 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] - BSA and Azide free (AB211045)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184718).

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

• WB

Unknown

Western blot - Anti-MLKL antibody [EPR17514] - BSA and Azide free (AB211045)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/mlkl-antibody-epr17514-ab184718'>ab184718</a>) 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='/en-us/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='/en-us/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

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