重组Anti-GRB2抗体[Y237]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y237] (AB32037)

Immunohistochemical analysis of GRB2 expression in paraffin embedded human ovarian carcinoma, using 1/100 ab32037.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y237] (AB32037)

Immunocytochemistry/immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilised HeLa (Human cervix adenocarcinoma epithelial cell) cells staining GRB2 with ab32037 at 1/200 dilution (green), followed by AlexaFluor®488 Goat anti-Rabbit secondary antibody (ab150077) at 1/1000 dilution (blue). Confocal image showing nuclear and weak cytoplasmic staining in HeLa cell line. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used as a counterstain at 1/200 dilution (red). DAPI was used as a nuclear counterstain.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is [secondary antibody name] ab150077 at 1/1000 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GRB2 antibody [Y237] (AB32037)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GRB2 (red) with ab32037 at a 1/20 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

Lab

Western blot - Anti-GRB2 antibody [Y237] (AB32037)

Anti-GRB2 antibody [Y237] (ab32037) staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32037 was shown to bind specifically to GRB2. A band was observed at 26 kDa in wild-type A431 cell lysates with a reduction in signal observed at this size in GRB2 heterozygous knockout cell line. To generate this image, wild-type and GRB2 heterozygous knockout A431 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-GRB2 antibody [Y237] (ab32037) at 1/5000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

Western blot - Human GRB2 heterozygous knockout A-431 cell line (<a href='/products/cell-lines/human-grb2-heterozygous-knockout-a-431-cell-line-ab276085'>ab276085</a>)

Lane 2:

GRB2 knockout A431 cell lysate at 20 µg

Lane 3:

HCT 116 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Secondary

Lanes 1 - 4:

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

Lanes 1 - 4:

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

Predicted band size: 25 kDa

Observed band size: 26 kDa

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• WB

Lab

Western blot - Anti-GRB2 antibody [Y237] (AB32037)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32037 observed at 25 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab32037 was shown to react with GRB2 in western blot. The band observed in the CRISPR-Cas9 edited cell lysate lane above 25 kDa is likely to represent an insertion. This has not been investigated further and the functional properties of the gene product have not been determined. Cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab32037 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-GRB2 antibody [Y237] (ab32037) at 1/5000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

GRB2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 3:

HL-60 (Human promyelocytic leukemia cell line) whole cell lysate at 20 µg

Lane 4:

Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 25 kDa

Observed band size: 25 kDa

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• WB

PubMed

Western blot - Anti-GRB2 antibody [Y237] (AB32037)

All lanes:

Western blot - Anti-GRB2 antibody [Y237] (ab32037) at 1/5000 dilution

All lanes:

Lysate prepared from human Mesothelial cell line MeT-5A at 4 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/2000 dilution

Predicted band size: 25 kDa

Observed band size: 24 kDa

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Exposure time: 1min

This image is a courtesy of Anonymous Abreview

• WB

Supplier Data

Western blot - Anti-GRB2 antibody [Y237] (AB32037)

Blocking/Dilution buffer : 5% NFDM/TBST

Exposure time : 3 minutes

All lanes:

Western blot - Anti-GRB2 antibody [Y237] (ab32037) at 1/10000 dilution

Lane 1:

PC-12 (rat adrenal gland pheochromocytoma) cell lysate at 10 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) cell lysate at 10 µg

Lane 3:

HEK293 (human epithelial cell line from embryonic kidney) cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/2000 dilution

Predicted band size: 25 kDa

Observed band size: 25 kDa

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• WB

Unknown

Western blot - Anti-GRB2 antibody [Y237] (AB32037)

All lanes:

Western blot - Anti-GRB2 antibody [Y237] (ab32037) at 1/5000 dilution

All lanes:

Western blot - Recombinant Human GRB2 protein (<a href='/products/proteins-peptides/recombinant-human-grb2-protein-ab85917'>ab85917</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: 25 kDa

true

Exposure time: 2min