Anti-CrkL antibody [Y244] - BSA and Azide free

• Flow Cyt

Lab

Flow Cytometry - Anti-CrkL antibody [Y244] - BSA and Azide free (AB247210)

This data was developed using ab32018, the same antibody clone in a different buffer formulation.
Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling CrkL with purified ab32018 at 1/20 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CrkL antibody [Y244] - BSA and Azide free (AB247210)

This data was developed using ab32018, the same antibody clone in a different buffer formulation.

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CrkL antibody [Y244] - BSA and Azide free (AB247210)

This data was developed using ab32018, the same antibody clone in a different buffer formulation.
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.

• IP

Lab

Immunoprecipitation - Anti-CrkL antibody [Y244] - BSA and Azide free (AB247210)

This data was developed using ab32018, the same antibody clone in a different buffer formulation.
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

All lanes:

Immunoprecipitation - Anti-CrkL antibody [Y244] (<a href='/en-us/products/primary-antibodies/crkl-antibody-y244-ab32018'>ab32018</a>)

Predicted band size: 34 kDa

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• WB

Lab

Western blot - Anti-CrkL antibody [Y244] - BSA and Azide free (AB247210)

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

All lanes:

Western blot - Anti-CrkL antibody [Y244] (<a href='/en-us/products/primary-antibodies/crkl-antibody-y244-ab32018'>ab32018</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CRKL knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CRKL knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-crkl-knockout-hela-cell-line-ab265993'>ab265993</a>)

Lane 3:

K-562 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 34 kDa

Observed band size: 37 kDa

false

• WB

Unknown

Western blot - Anti-CrkL antibody [Y244] - BSA and Azide free (AB247210)

All lanes:

Western blot - Anti-CrkL antibody [Y244] (<a href='/en-us/products/primary-antibodies/crkl-antibody-y244-ab32018'>ab32018</a>) at 1/1000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 15 µg

Lane 2:

Mouse thymus lysate at 15 µg

Lane 3:

Rat thymus lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 34 kDa

false