Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free

• WB

Unknown

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free (AB240399)

This data was developed using ab222320, the same antibody clone in a different buffer formulation.

Blocking/Dilution buffer : 5% NFDM/TBST.

The higher MW bands in Lanes 1 and 2 are extra bands.

The lysates were prepared following the 1% SDS Hot Lysate buffer method.

For Lysate preparation protocol, please refer to the protocol section of the website and/or here.

All lanes:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free (ab240399) at 1/1000 dilution

Lane 1:

Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 10 µg

Lane 2:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours at 10 µg

Lane 3:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours, then treated with alkaline phosphatase for 1 hour 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 1/20000 dilution

Predicted band size: 57 kDa

Observed band size: 53 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free (AB240399)

This data was developed using ab222320, the same antibody clone in a different buffer formulation.

Blocking/Dilution buffer : 5% NFDM/TBST.

Cells were lysed in 4% SDS buffer (4% SDS,10% 2-ME, 20% glycerol, 0.004% Bromophenol blue, Tris-HCl).

The images were kindly provided by our collaborator Dr. Jiahuai Han, Xiamen University.

All lanes:

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free (ab240399) at 1/500 dilution

Lane 1:

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

Lane 2:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 10 ng/ml TNF-a and 20 µM z-VAD for 2 hours at 20 µg

Lane 3:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate treated with 10 ng/ml TNF-a and 20 µM z-VAD for 3.5 hours at 20 µg

Lane 4:

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

Lane 5:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate with RIP3 KO cells treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hours 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 1/5000 dilution

Predicted band size: 57 kDa

false

Exposure time: 60s

• WB

Supplier Data

Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free (AB240399)

This data was developed using ab222320, 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

Lab

Dot Blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] - BSA and Azide free (AB240399)

Dot blot analysis of RIP3 (phospho T231 + S232) labeled with ab222320 at 1/1000 dilution.

Lane 1 : RIP3 (phospho T231 + S232) peptide (aa229-239);

Lane 2 : RIP3 (phospho T231 + S232) peptide (aa223-236);

Lane 3 : RIP3 (phospho T231) peptide (aa223-239);

Lane 4 : RIP3 (phospho S232) peptide (aa223-239);

Lane 5 : RIP3 non-phospho peptide (aa223-239).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab222320).