Anti-RIP antibody [EPR24883-85]

• IHC

Supplier Data

Immunohistochemistry - Anti-RIP antibody [EPR24883-85] (AB300617)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 c tissue labeling RIP with ab300617 at 1/100 (5.11 ug/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on (A) Wild-type HAP1 cell pellet(B) and no staining on RIPK1 knockout HAP1 cell pellet.The section was incubated with ab300617 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC

Supplier Data

Immunohistochemistry - Anti-RIP antibody [EPR24883-85] (AB300617)

Immunohistochemical analysis of paraffin-embedded Human cervical carci tissue labeling RIP with ab300617 at 1/100 (5.11 ug/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Cytoplasmic staining on human cervical carcinoma.The section was incubated with ab300617 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

Lab

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

Anti-RIPK1 antibody [EPR24883-85] (ab300617) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab300617 was shown to bind specifically to RIPK1. A band was observed at 75 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line ab276121 (knockout cell lysate ab284221). To generate this image, wild-type and RIPK1 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween$®$ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

All lanes:

Western blot - Anti-RIP antibody [EPR24883-85] (ab300617) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

RIPK1 knockout THP-1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Predicted band size: 75 kDa

Observed band size: 75 kDa

false

• WB

Supplier Data

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

The expression of RIP (phospho S166) is upregulated in response to z-VAD, TNF alpha and SM-164, while it is downregulated in response to necrostain treatment (PMID : 32027418, PMID : 33273695).

The identity of the lower MW band at approximately 35 kDa is unknown.

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

In Western blot, Anti-RIP antibody - Total protein control (ab300617) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-RIP (phospho S166) antibody [EPR25654-166] (<a href='/en-us/products/primary-antibodies/rip-phospho-s166-antibody-epr25654-166-ab316923'>ab316923</a>) at 1/1000 dilution

Lane 1:

Untreated HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 40 µg with NFDM/TBST

Lane 2:

HT-29 treated with 20 uM z-VAD for 3.5 hours, then with 20 ng/ml TNF alpha, 100 nM SM-164 and 50 uM necrostatin added for 3 hours whole cell lysate (untreated membrane) at 40 µg with NFDM/TBST

Lane 3:

HT-29 treated with 20 uM z-VAD for 3.5 hours, then with 20 ng/ml TNF alpha and 100nM SM-164 added for 3 hours whole cell lysate (untreated membrane) at 40 µg with NFDM/TBST

Lane 4:

HT-29 treated with 20 uM z-VAD for 3.5 hours, then with 20 ng/ml TNF alpha and 100 nM SM-164 added for 3 hours whole cell lysate (Lambda phosphatase treated membrane) at 40 µg with NFDM/TBST

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: 75 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

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

In Western blot, ab316923 was shown to bind specifically to RIP. Target of interest was observed at 75 kDa in wild-type HAP1 cell lysates (lanes 1 and 3), whereas no signal observed at this size in RIP knockout cell line lysates (lanes 2 and 4).

The expression of RIP (phospho S166) is upregulated in response to z-VAD, TNF alpha and SM-164 treatment (PMID : 32027418, PMID : 33273695).

The identity of the lower MW band at approximately 15 kDa is unknown.

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

In Western blot, Anti-RIP antibody - Total protein control (ab300617) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-RIP (phospho S166) antibody [EPR25654-166] (<a href='/en-us/products/primary-antibodies/rip-phospho-s166-antibody-epr25654-166-ab316923'>ab316923</a>) at 1/1000 dilution

Lane 1:

Untreated wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 50 µg

Lane 2:

Untreated RIP knockout HAP1 whole cell lysate at 50 µg

Lane 3:

Wild-type HAP1 treated with 20 uM z-VAD for 3.5 hours, then with 20 ng/ml TNF alpha and 100 nM SM-164 added for 3 hours whole cell lysate at 50 µg

Lane 4:

RIP knockout HAP1 treated with 20 uM z-VAD for 3.5 hours, then with 20 ng/ml TNF alpha and 100 nM SM-164 added for 3 hours whole cell lysate at 50 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: 75 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

All lanes:

Western blot - Anti-RIP antibody [EPR24883-85] (ab300617) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Rat testis tissue 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

false

• WB

Supplier Data

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

The identity of the lower MW band at approximately 35 kDa is unknown.

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

In Western blot, Anti-RIP antibody - Total protein control (ab300617) staining at 1/1000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-RIP (phospho S166) antibody [EPR25654-166] (<a href='/en-us/products/primary-antibodies/rip-phospho-s166-antibody-epr25654-166-ab316923'>ab316923</a>) at 1/1000 dilution

Lane 1:

Untreated 293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg with NFDM/TBST

Lane 2:

Untreated 293T transfected with a human wild-type RIP expression vector containing a myc-His-tag®, whole cell lysate at 20 µg with NFDM/TBST

Lane 3:

293T transfected with a human wild-type RIP expression vector containing a myc-His-tag® were treated with 100 nM Calyculin for 30 minutes, whole cell lysate at 20 µg with NFDM/TBST

Lane 4:

293T transfected with a human RIP (S166A mutation) expression vector containing a myc-His-tag® were treated with 100 nM Calyculin for 30 minutes, whole cell lysate at 20 µg with NFDM/TBST

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: 75 kDa,36 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

All lanes:

Western blot - Anti-RIP antibody [EPR24883-85] (ab300617) at 1/1000 dilution

All lanes:

Mouse liver tissue lysate at 20 µg

Secondary

Lanes 1 - 3:

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 1/10000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

false

• WB

Supplier Data

Western blot - Anti-RIP antibody [EPR24883-85] (AB300617)

All lanes:

Western blot - Anti-RIP antibody [EPR24883-85] (ab300617) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma) 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

false