Recombinant Anti-CrkL antibody [Y244] (ab32018)

Lanes 1-3: Merged signal (red and green). Green - ab32018 observed at 37 kDa. Red - loading control ab7291 observed at 50 kDa.

ab32018 Anti-CrkL antibody [Y244] was shown to specifically react with CrkL in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265993 (knockout cell lysate ab257397) was used. Wild-type and CrkL knockout samples were subjected to SDS-PAGE. ab32018 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue sections labeling CrkL with purified ab32018 at 1/100 dilution (2.24 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunocytochemistry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling CrkL with purified ab32018 at 1/50 dilution (4.5 µg/mL). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Purified ab32018 at 1/50 dilution (2µg) immunoprecipitating CrkL in K-562 whole cell lysate.
Lane 1 (input): K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10µg
Lane 2 (+): ab32018 + K-562 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32018 in K-562 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 37 kDa