重组Anti-GRB2抗体[Y301] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y301] - BSA and Azide free (AB247231)

This data was developed using ab32111, the same antibody clone in a different buffer formulation.

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GRB2 antibody [Y301] - BSA and Azide free (AB247231)

This data was developed using ab32111, the same antibody clone in a different buffer formulation.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 >5,000 events was performed.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y301] - BSA and Azide free (AB247231)

This data was developed using ab32111, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of GRB2 expression in PC12 cells, using 1/100 ab32111.

• WB

Lab

Western blot - Anti-GRB2 antibody [Y301] - BSA and Azide free (AB247231)

This data was developed using ab32111, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-GRB2 antibody [Y301] (<a href='/products/primary-antibodies/grb2-antibody-y301-ab32111'>ab32111</a>) 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] - BSA and Azide free (AB247231)

This data was developed using ab32111, the same antibody clone in a different buffer formulation. 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] (<a href='/products/primary-antibodies/grb2-antibody-y301-ab32111'>ab32111</a>) 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