重组Anti-Eph receptor B2抗体[EPR22422-67]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Eph receptor B2 antibody [EPR22422-67] (AB307811)

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized COLO 205 (human colon adenocarcinoma tumor ascetic epithelial cell) cells labelling Eph receptor B2 with ab307811 at 1/1000 (0.518 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preabsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing membranous staining in COLO 205 cell line. Negative control : K-562. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preabsorbed at 1/1000 2ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Eph receptor B2 antibody [EPR22422-67] (AB307811)

Eph receptor B2 was immunoprecipitated from 0.35 mg COLO 205 (human colorectal adenocarcinoma cell) whole cell lysate 10 ug with ab307811 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307811 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : COLO 205 (human colorectal adenocarcinoma cell) whole cell lysate 10 ug Lane 2 : ab307811 IP in COLO 205 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307811 in COLO 205 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

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Immunoprecipitation - Anti-Eph receptor B2 antibody [EPR22422-67] (ab307811) at 1/1000 dilution

All lanes:

COLO 205 cells

Secondary

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Immunoprecipitation at 1/5000 dilution

Observed band size: 130 kDa

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Exposure time: 3s

• WB

Lab

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (AB307811)

Western blot : Anti-EPHB2 antibody [EPR22422-67] (ab307811) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab307811 was shown to bind specifically to EPHB2. A band was observed at 130 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EPHB2 knockout cell line. To generate this image, wild-type and EPHB2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Eph receptor B2 antibody [EPR22422-67] (ab307811) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EPHB2 knockout HCT 116 cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 130 kDa

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• WB

Supplier Data

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (AB307811)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody does not cross-react with human EPHB1 and EPHB3. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 6 seconds

All lanes:

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (ab307811) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a myc-his tag whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human EPHB2 expression vector containing a myc-his tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human EPHB1 expression vector containing a myc-his tag whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human EPHB3 expression vector containing a myc-his tag whole cell lysate 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 1/100000 dilution

Observed band size: 130 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (AB307811)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : K562 The lower band around 70 kDa could be non-specific band or cleaved fragment (PMID : 18713744, PMID : 21508957). Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : Lane 1 : 125 seconds, Lane 2 and 3 : 48 seconds

All lanes:

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (ab307811) at 1/1000 dilution

Lane 1:

HT-1080 (human fibrosarcoma epithelial cell)whole cell lysate at 50 µg

Lane 2:

COLO 205 (human colon adenocarcinoma tumor ascetic epithelial cell) whole cell lysate at 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 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 1/100000 dilution

Observed band size: 130 kDa

false

• WB

Supplier Data

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (AB307811)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lane 3 was freshly made and used immediately to minimize protein degradation. The lower band around 80 kDa could be non-specific band or cleaved fragment (PMID : 18713744). Lanes 1 and 2 are non-boiled as boiling may cause protein aggregation. Exposure time : Lanes 1 and 2 : 136 seconds, Lane 3 : 180 seconds

All lanes:

Western blot - Anti-Eph receptor B2 antibody [EPR22422-67] (ab307811) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 40 µg

Lane 2:

Rat brain tissue lysate at 40 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 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 1/100000 dilution

Observed band size: 130 kDa

false