Anti-RAC1 + Cdc42 (phospho S71) antibody

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAC1 + Cdc42 (phospho S71) antibody (AB5482)

Immunohistochemiscal analysis of paraffin-embedded human skin tissue labeling RAC1 + Cdc42 (phospho S71) with ab5482 at 1/100 dilution (right) compared to a negative control without primary antibody (left).

To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab5482 diluted in 3% BSA-PBS at a dilution of 1/100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAC1 + Cdc42 (phospho S71) antibody (AB5482)

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling RAC1 + Cdc42 (phospho S71) with ab5482 at 1/20 dilution (right) compared to a negative control without primary antibody (left).

To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab5482 diluted in 3% BSA-PBS at a dilution of 1/20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• WB

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Western blot - Anti-RAC1 + Cdc42 (phospho S71) antibody (AB5482)

Peptide Competition and Phosphatase Treatment :
Lysates prepared from A431 cells left unstimulated (1) or stimulated with EGF (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either not treated (1-5) or treated with lambda phosphatase (6), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab5482 antibody for two hours at room temperature in a 3% BSATBST buffer, following prior incubation with : no peptide (1, 2, 6), the nonphosphopeptide corresponding to the immunogen (3), a generic phosphoserine containing peptide (4), or, the phosphopeptide immunogen (5), After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that the peptide corresponding to ab5482 blocks the antibody signal. The data also shows that phosphatase stripping eliminates the signal, verifying that the antibody is pho

All lanes:

Western blot - Anti-RAC1 + Cdc42 (phospho S71) antibody (ab5482)

Predicted band size: 21 kDa

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