Anti-RILPL2 antibody [EPR29763-69] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-RILPL2 antibody [EPR29763-69] - BSA and Azide free (AB326586)

This data was developed using ab325436, the same antibody clone in a different buffer formulation.

RILPL2 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab325436 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325436 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 2 : ab325436 at 1/30 IP in THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325436 in THP-1 whole cell lysate

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

Exposure time : 137 seconds.

All lanes:

Immunoprecipitation - Anti-RILPL2 antibody [MJF-D29763-69] (<a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a>) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a> at 1/30 IP in THP-1 (human monocytic leukemia monocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a> in THP-1 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 26 kDa

false

Exposure time: 137s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RILPL2 antibody [EPR29763-69] - BSA and Azide free (AB326586)

This data was developed using ab325436, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized EL4 (mouse lymphoma T lymphocyte) and Neuro-2a (mouse neuroblastoma neuroblast) cells labelling RILPL2 with ab325436 at 1/50 (10.34 µg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in EL4 cell line and no staining in Neuro-2a cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).

Low expression : Neuro-2a.

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

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab325436 at a 1/50 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab7291 at a 1/1000 dilution followed by ab150081 at a 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-RILPL2 antibody [EPR29763-69] - BSA and Azide free (AB326586)

This data was developed using ab325436, the same antibody clone in a different buffer formulation.

RILPL2 was immunoprecipitated from 0.35 mg EL4 (mouse lymphoma T lymphocyte) whole cell lysate with ab325436 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325436 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : EL4 (mouse lymphoma T lymphocyte) whole cell lysate
Lane 2 : ab325436 at 1/30 IP in EL4 (mouse lymphoma T lymphocyte) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325436 in EL4 whole cell lysate

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

Exposure time : 137 seconds.

All lanes:

Immunoprecipitation - Anti-RILPL2 antibody [MJF-D29763-69] (<a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a>) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a> at 1/30 IP in EL4 (mouse lymphoma T lymphocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a> in EL4 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 22 kDa

false

Exposure time: 137s

• WB

Lab

Western blot - Anti-RILPL2 antibody [EPR29763-69] - BSA and Azide free (AB326586)

This data was developed using ab325436, the same antibody clone in a different buffer formulation.

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

To minimize protein degradation lane 2 was lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

Lane2 was developed using a high-sensitivity ECL substrate allowing for the detection of proteins in the mid-femtogram range.

Lane1 is incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and lane 2 is incubated with Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated (ab97051) at 1/100000.

In Western blot Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-RILPL2 antibody [MJF-D29763-69] (<a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a>) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 40 µg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lane 1:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lane 2:

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: 26 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-RILPL2 antibody [EPR29763-69] - BSA and Azide free (AB326586)

This data was developed using ab325436, the same antibody clone in a different buffer formulation.

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

Low expression : SK-BR-3(PMID : 31497344) A549

This blot was developed using a high-sensitivity ECL substrate allowing for the detection of proteins in the mid-femtogram range.

In Western blot Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-RILPL2 antibody [MJF-D29763-69] (<a href='/en-us/products/primary-antibodies/rilpl2-antibody-mjf-d29763-69-ab325436'>ab325436</a>) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 40 µg

Lane 2:

U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 40 µg

Lane 3:

SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

A549 (human lung carcinoma epithelial cell) whole cell 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: 26 kDa,36 kDa

true

Exposure time: 180s