Anti-GRB2 antibody [Y237] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GRB2 antibody [Y237] - BSA and Azide free (AB227117)

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 Fluor^® 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32037).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y237] - BSA and Azide free (AB227117)

This IHC data was generated using the same anti-GRB2 antibody clone, Y237, in a different buffer formulation (cat# 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

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y237] - BSA and Azide free (AB227117)

Immunofluorescent analysis of GRB2 expression in HeLa cells using 1/50 ab32037.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32037).

• WB

Lab

Western blot - Anti-GRB2 antibody [Y237] - BSA and Azide free (AB227117)

This data was developed using ab32037, the same antibody clone in a different buffer formulation.

Western blot : Anti-GRB2 antibody [Y237] ab32037 staining at 1/2000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 27 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in GRB2 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-GRB2 antibody [Y237] (<a href='/ko/products/primary-antibodies/grb2-antibody-y237-ab32037'>ab32037</a>) at 1/2000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human GRB2 knockout HCT116 cell line (<a href='/ko/products/cell-lines/human-grb2-knockout-hct116-cell-line-ab273715'>ab273715</a>) at 20 µg

Lane 3:

A431 at 20 µg

Lane 4:

HeLa at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 27 kDa

Observed band size: 27 kDa

false

• WB

Lab

Western blot - Anti-GRB2 antibody [Y237] - BSA and Azide free (AB227117)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/ko/products/primary-antibodies/grb2-antibody-y237-ab32037'>ab32037</a>) 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

false

• WB

Lab

Western blot - Anti-GRB2 antibody [Y237] - BSA and Azide free (AB227117)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/ko/products/primary-antibodies/grb2-antibody-y237-ab32037'>ab32037</a>) 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='/ko/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

false