重组Anti-Ras抗体[EP1125Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ras antibody [EP1125Y] (AB52939)

Immunocytochemistry analysis of MCF7 (human breast adenocarcinoma cell line) cells labeling Ras with Purified ab52939 at 1 : 500 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor ® 594) 1 : 200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor ® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Ras antibody [EP1125Y] (AB52939)

ab52939 staining of GTPase KRas in a HCT 116 cell spheroid. The cells were fixed with 4% paraformaldehyde (10 min), permeabilised with 2% Triton X-100 for 1h and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween overnight at room temperature. The spheroids were then incubated overnight at room temperature with ab52939 at 2 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 2 μg/ml. DAPI was used as nuclear counterstain (shown in blue). As secondary antibodies ab150081 Goat anti-Rabbit (Alexa Fluor^® 488) (shown in green) and ab150120 Goat anti-Mouse (Alexa Fluor^® 594) (shown in magenta) were used, incubated overnight at room temperature. All permeabilization, blocking and antibody incubation steps were performed using a rotary shaker.

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Similar results were observed using 100% Methanol (5min).

• IP

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Immunoprecipitation - Anti-Ras antibody [EP1125Y] (AB52939)

ab52939 (purified) at 1 : 20 dilution (2μg) immunoprecipitating Ras in Jurkat whole cell lysate.

Lane 1 (input) : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate 10μg
Lane 2 (+) : ab52939 & Jurkat whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab52939 in Jurkat whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Ras antibody [EP1125Y] (ab52939)

Predicted band size: 21 kDa

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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ras antibody [EP1125Y] (AB52939)

Intracellular Flow Cytometry analysis of PC-12 (rat adrenal gland pheochromocytoma cell line) cells labeling Ras with purified ab52939 at 1/30 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ras antibody [EP1125Y] (AB52939)

Unpurified ab52939 at 1/100 dilution staining Ras in permeabilized PC-12 (rat adrenal gland pheochromocytoma cell line) cells by intracellular flow cytometry (red). Rabbit IgG negative control (green).

• WB

Lab

Western blot - Anti-Ras antibody [EP1125Y] (AB52939)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Ras antibody [EP1125Y] (ab52939) at 1/5000 dilution

Lane 1:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysates at 20 µg

Lane 2:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysates at 20 µg

Lane 3:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysates at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma cell line) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-Ras antibody [EP1125Y] (AB52939)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Ras antibody [EP1125Y] (ab52939) at 1/5000 dilution

All lanes:

PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 21 kDa

false

• WB

Unknown

Western blot - Anti-Ras antibody [EP1125Y] (AB52939)

All lanes:

Western blot - Anti-Ras antibody [EP1125Y] (ab52939) at 1/500000 dilution

All lanes:

C6 (rat glial tumor cell line) cell lysate at 10 µg/mL

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 21 kDa

Observed band size: 18 kDa

false

• WB

Unknown

Western blot - Anti-Ras antibody [EP1125Y] (AB52939)

All lanes:

Western blot - Anti-Ras antibody [EP1125Y] (ab52939) at 1/500 dilution

All lanes:

Human Ras full length protein (<a href='/products/unavailable/human-ras-full-length-protein-ab56526'>ab56526</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 21 kDa

true

Exposure time: 1min