Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910)

MLKL (phospho S345) was immunoprecipitated from 1mg of L-929 (Mouse connective tissue fibroblast cells) whole cell lysate treated with 20 ng/ml TNF alpha + 100 nM Smac mimetic + 20 µM z-VAD compound for 8h using ab196436 at 1/150 dilution. Western blot was performed from the immunoprecipitate using ab196436 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : L-929 whole cell lysate treated with 20 ng/ml TNF alpha+ 100 nM Smac mimetic+ 20 µM z-VAD compound for 8h;10 µg (Input).
Lane 2 : ab196436 IP in L-929 whole cell lysate treated with 20 ng/ml TNF alpha+ 100 nM Smac mimetic+ 20 µM z-VAD compound for 8h.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab196436 in L-929 whole cell lysate treated with 20 ng/ml TNF alpha+ 100 nM Smac mimetic+ 20 µM z-VAD compound for 8h.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab196436).

All lanes:

Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (<a href='/en-us/products/primary-antibodies/mlkl-phospho-s345-antibody-epr95152-ab196436'>ab196436</a>)

All lanes:

Immunoprecipitation - Recombinant human TNF alpha protein (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-ab9642'>ab9642</a>)

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab196436).

Blocking and diluting buffer : 5% NFDM/TBTS.

ab181602 was used as a loading control at 1/1000000 dilution.

We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.

Lanes 1 - 4:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [2D7] (<a href='/en-us/products/primary-antibodies/rip3-phospho-t231-s232-antibody-2d7-ab205421'>ab205421</a>) at 1/1000 dilution

Lanes 5 - 8:

Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (<a href='/en-us/products/primary-antibodies/mlkl-phospho-s345-antibody-epr95152-ab196436'>ab196436</a>) at 1/1000 dilution

Lanes 1, 3, 5 and 7:

Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

Lanes 2 and 6:

L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysate at 20 µg

Lanes 4 and 8:

L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/20000 dilution

Lanes 5 - 8:

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: 57 kDa,54 kDa

Observed band size: 53 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910)

This data was developed using ab196436, the same antibody clone in a different buffer formulation.

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

ab181602 GAPDH was used as a loading control at 1/1000000 dilution.

We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.

Lanes 1 - 4:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (<a href='/en-us/products/primary-antibodies/rip3-phospho-t231-s232-antibody-epr19403-52-ab222320'>ab222320</a>) at 1/1000 dilution

Lanes 5 - 8:

Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (<a href='/en-us/products/primary-antibodies/mlkl-phospho-s345-antibody-epr95152-ab196436'>ab196436</a>) at 1/1000 dilution

Lanes 1, 3, 5 and 7:

Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

Lanes 2 and 6:

L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysate at 20 µg

Lanes 4 and 8:

L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours 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: 57 kDa,54 kDa

Observed band size: 53 kDa

false

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910)

This Dot Blot data was generated using the same anti-MLKL (phospho S345) antibody clone, EPR9515(2), in a different buffer formulation (cat# ab196436).

Dot blot analysis of MLKL (phospho S345) peptide (Lane 1), and non-phospho peptide (Lane 2), labeled using ab196436 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.