Anti-RILPL1 抗体 [MJF-R41-21] (ab302492)

All lanes : Anti-RILPL1 antibody [MJF-R41-21] (ab302492) at 1/1000 dilution

Lane 1 : Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 2 : RILPL1 knockout A549 whole cell lysate
Lane 3 : HeLa (human cervical adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 42,47 kDa why is the actual band size different from the predicted?

Blocking / Diluting buffer and: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

False colour image of Western blot: Anti-RILPL1 antibody [MJF-R41-21] (ab302492) 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, ab302492 was shown to bind specifically to RILPL1. Two bands were observed at 47/42 kDa in wild-type A549 cell lysates whereas no signal observed at this size in RILPL1 knockout cell line. To generate this image, wild-type and RILPL1 knockout A549 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel and then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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. 

The doublet bands represent two isoforms of RILPL1.

Wild-type and RILPL1 knockout A549 cell lysates were kindly provided by Dr. Dario Alessi, University of Dundee.

All lanes : Anti-RILPL1 antibody [MJF-R41-21] (ab302492) at 1/1000 dilution

Lane 1 : Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 2 : RILPL1 knockout A549 whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 4 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 5 : Rat brain tissue lysate
Lane 6 : Rat kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 42,47 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking / Diluting buffer and: 5% NFDM/TBST.

The doublet bands represent two isoforms of RILPL1.

Negative control: kidney (PMID: 14668488).

Wild-type and RILPL1 knockout A549 cell lysates were kindly provided by Dr. Dario Alessi, University of Dundee.

All lanes : Anti-RILPL1 antibody [MJF-R41-21] (ab302492) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse kidney tissue lysate
Lane 3 : Mouse liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/20000 dilution

Predicted band size: 47 kDa
Observed band size: 47 kDa


Exposure time: 103 seconds

Blocking / Diluting buffer and: 5% NFDM/TBST.

Negative control: kidney, liver (PMID: 14668488).

All lanes : Anti-RILPL1 antibody [MJF-R41-21] (ab302492) at 1/1000 dilution

Lane 1 : SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate
Lane 2 : U937 (human histiocytic lymphoma monocyte), whole cell lysate
Lane 3 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate
Lane 4 : Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 42,47 kDa why is the actual band size different from the predicted?


Exposure time: 37 seconds

Blocking / Diluting buffer and: 5% NFDM/TBST.

The doublet bands represent two isoforms of RILPL1.
Low expression: U937 (Human Protein Atlas).

All lanes : Anti-RILPL1 antibody [MJF-R41-21] (ab302492) at 1/1000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human hypothalamus tissue lysate
Lane 3 : Human heart tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 47 kDa
Observed band size: 42,47 kDa why is the actual band size different from the predicted?


Exposure time: 26 seconds

Blocking / Diluting buffer and: 5% NFDM/TBST.

The doublet bands represent two isoforms of RILPL1.

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) labelling RILPL1 with ab302492 at 1/250 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Confocal image showing centrosome and cytoplasmic staining in SH-SY5Y cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) at 1/200 dilution.

Low expression: U-937

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) labelling RILPL1 with ab302492 at 1/250 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Confocal image showing centrosome staining in Neuro-2a cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) at 1/200 dilution.

RILPL1 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell), whole cell lysate with ab302492 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302492 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (Input): A549 whole cell lysate 10 μg

Lane 2 (+): A549 whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab302492 in A549 whole cell lysate

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

RILPL1 was immunoprecipitated from 0.35 mg mouse brain tissue lysate with ab302492 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302492 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (Input): Mouse brain tissue lysate 10 μg

Lane 2 (+): Mouse brain tissue lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab302492 in mouse brain tissue lysate

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

Exposure time: 3 minutes.

RILPL1 was immunoprecipitated from 0.35 mg rat brain tissue lysate with ab302492 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302492 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (Input): Rat brain tissue lysate 10 μg

Lane 2 (+): Rat brain tissue lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab302492 in rat brain tissue lysate

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.