Anti-GRB2 抗体 [Y301]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GRB2 antibody [Y301] (AB32111)

Overlay histogram showing SH-SY5Y cells stained with ab32111 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32111 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5000 events was performed.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y301] (AB32111)

Immunohistochemical analysis of GRB2 expression in paraffin embedded human breast carcinoma tissue section using 1/100 ab32111. Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y301] (AB32111)

Immunofluorescent analysis of GRB2 expression in PC12 cells, using 1/100 ab32111.

• WB

Unknown

Western blot - Anti-GRB2 antibody [Y301] (AB32111)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32111 observed at 25 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32111 was shown to react with GRB2 in wild-type HCT 116 cells in western blot with loss of signal observed in GRB2 knockout cell line ab273715 (GRB2 knockout cell lysate ab275248). Wild-type and GRB2 knockout HCT 116 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab32111 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GRB2 antibody [Y301] (ab32111) at 1/2000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

GRB2 knockout HCT116 cell lysate at 20 µg

Lane 2:

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

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HEK293 cell lysate at 20 µg

Predicted band size: 25 kDa

Observed band size: 25 kDa

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• WB

Supplier Data

Western blot - Anti-GRB2 antibody [Y301] (AB32111)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody recognises GRB2 isoform 1 (ASH-L) and not GRB2 isoform 2 (GRB3-3).

All lanes:

Western blot - Anti-GRB2 antibody [EPR24634-17] (<a href='/products/primary-antibodies/grb2-antibody-epr24634-17-ab315005'>ab315005</a>) at 1/1000 dilution

Lane 1:

Human GRB2 isoform 1 recombinant protein at 10 ng

Lane 2:

Human GRB2 isoform 2 recombinant protein at 10 ng

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: 25 kDa,60 kDa

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Exposure time: 92s

• WB

Lab

Western blot - Anti-GRB2 antibody [Y301] (AB32111)

Blocking/Diluting Buffer and concentration : 5% NFDM/TBST

We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-GRB2 antibody [Y301] (ab32111) at 1/5000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 15 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

Lane 4:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 15 µg

Lane 5:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 15 µg

Lane 6:

C6 (Rat glial tumor glial cell) whole cell lysate 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: 25 kDa

Observed band size: 25 kDa

false

• WB

Lab

Western blot - Anti-GRB2 antibody [Y301] (AB32111)

Anti-GRB2 antibody [Y301] (ab32111) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32111 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 [Y301] (ab32111) at 1/1000 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

false