Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse T cell lymphoma tissue labeling RIP3 with ab322555 at 1/500 (1.064 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse T cell lymphoma. The section was incubated with ab322555 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized L-929 (mouse connective tissue fibroblast) cells labelling RIP3 with ab322555 at 1/500 (1.064 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in L-929 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : Neuro-2a.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling RIP3 with ab322555 at 1/500 (1.064 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon. The section was incubated with ab322555 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling RIP3 with ab322555 at 1/500 (1.064 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : weak staining on mouse liver. The section was incubated with ab322555 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• WB

Supplier Data

Western blot - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

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

Negative control : Neuro-2a B16-F10 (PMID : 25858093).

The molecular weight observed is consistent with what has been described in the literature (PMID : 38514849). The ~38 kDa band likely represents a N-terminal cleavage product of RIP3.

This antibody was tested on frozen and fresh L-929 Neuro-2a and J774A.1 whole cell lysates and it was concluded that RIP3 is the easy target for degradation.

To minimize protein degradation cells (lanes 6-8) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

Exposure time : Lanes 1-2 6-8 : 26 seconds; Lanes 3-5 : 59 seconds

All lanes:

Western blot - Anti-RIP3 antibody [EPR26080-567] (<a href='/en-us/products/primary-antibodies/rip3-antibody-epr26080-567-ab322555'>ab322555</a>) at 1/1000 dilution

Lanes 1 and 6:

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

Lanes 2 and 7:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 3:

J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 5:

B16-F10 (mouse skin melanoma cell) whole cell lysate at 20 µg

Lane 8:

J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) 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 1/100000 dilution

Observed band size: 53 kDa,38 kDa,15 kDa

false

• WB

Supplier Data

Western blot - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

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

Negative control : Neuro-2a.

The expression of RIP3 (phospho T231 + S232) is upregulated in response to z-VAD, TNF alpha and SM-164 treatment.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-RIP3 antibody (ab322555) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-RIP3 (phospho T231) antibody [EPR29676-66] (<a href='/en-us/products/primary-antibodies/rip3-phospho-t231-antibody-epr29676-66-ab324983'>ab324983</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

L-929 treated with 20 µM z-VAD for 0.5h, 20ng/ml TNF alpha and 100nM SM-164 was then added for additional 7h whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) treated with 20 µM z-VAD for 0.5h, 20ng/ml TNF alpha and 100nM SM-164 was then added for additional 7h whole cell lysate (untreated membrane) at 20 µg

Lane 5:

Untreated L-929 (mouse connective tissue fibroblast) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 6:

L-929 treated with 20 µM z-VAD for 0.5h, 20ng/ml TNF alpha and 100nM SM-164 was then added for additional 7h whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 7:

Untreated Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 8:

Neuro-2a (mouse neuroblastoma neuroblast) treated with 20 µM z-VAD for 0.5h, 20ng/ml TNF alpha and 100nM SM-164 was then added for additional 7h whole cell lysate (alkaline phosphatase treated membrane) 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

Observed band size: 50 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-RIP3 antibody [EPR26080-567] - BSA and Azide free (AB325961)

This data was developed using ab322555, the same antibody clone in a different buffer formulation.

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

Low expression : liver (PMID : 25950489).

The molecular weight observed is consistent with what has been described in the literature (PMID : 38514849). The ~38 kDa band likely represents a N-terminal cleavage product of RIP3.

This antibody was tested on frozen and fresh mouse colon and mouse spleen tissue lysates and it was concluded that RIP3 is the easy target for degradation.

To minimize protein degradation tissues (lanes 4-6) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

Exposure time : Lanes 1-4 : 180 seconds; Lane 5 : 37 seconds; Lane 6 : 70 seconds

All lanes:

Western blot - Anti-RIP3 antibody [EPR26080-567] (<a href='/en-us/products/primary-antibodies/rip3-antibody-epr26080-567-ab322555'>ab322555</a>) at 1/1000 dilution

Lane 1:

Mouse colon tissue lysate at 20 µg

Lane 2:

Mouse liver lysate at 20 µg

Lanes 3 and 6:

Mouse spleen tissue lysate at 20 µg

Lane 4:

Mouse testis tissue lysate at 40 µg

Lane 5:

Mouse colon tissue lysate at 40 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 1/100000 dilution

Observed band size: 53 kDa,38 kDa,15 kDa

false