Anti-CAD antibody [EP710Y] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.Unpurified ab40800 (1/100), staining human CAD (N-term) in HeLa cells by Immunofluorescence.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling CAD (red) with ab40800 at a 1/120 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.

Immunofluorescence staining of HeLa cells with purified ab40800 at a working dilution of 1/300, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab40800 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• IP

Unknown

Immunoprecipitation - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.ab40800 (purified) at 1/30 immunoprecipitating CAD in 10 μg HeLa (Lanes 1 and 2, observed at 250 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP (ab131366) was used for detection (1/10 000). Blocking buffer and concentration : 5% NFDM/TBST Dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-CAD antibody [EP710Y] (<a href='/en-us/products/primary-antibodies/cad-antibody-ep710y-ab40800'>ab40800</a>)

Predicted band size: 243 kDa

false

• WB

Lab

Western blot - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab40765 works better than ab40800 in western blot testing.

We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results when using ab955 in western blot.

Lanes 1 and 3:

Western blot - Anti-CAD antibody [EP710Y] (<a href='/en-us/products/primary-antibodies/cad-antibody-ep710y-ab40800'>ab40800</a>) at 1/1000 dilution

Lanes 2 and 4:

Western blot - Anti-CAD antibody [EP711Y] (<a href='/en-us/products/primary-antibodies/cad-antibody-ep711y-ab40765'>ab40765</a>) at 1/1000 dilution

Lanes 1 - 2:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 3 - 4:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 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: 243 kDa

Observed band size: 250 kDa

false

Exposure time: 10s

• WB

Unknown

Western blot - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-CAD antibody [EP710Y] (<a href='/en-us/products/primary-antibodies/cad-antibody-ep710y-ab40800'>ab40800</a>) at 1/1000 dilution

All lanes:

Western blot - HEK-293 whole cell lysate (<a href='/en-us/products/cell-lysates/hek-293-whole-cell-lysate-ab7902'>ab7902</a>) at 10 µg

Predicted band size: 243 kDa

Observed band size: 250 kDa

false

• WB

Lab

Western blot - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CAD antibody [EP710Y] (<a href='/en-us/products/primary-antibodies/cad-antibody-ep710y-ab40800'>ab40800</a>) at 1/1000 dilution

All lanes:

HeLa cell lysate at 1/20000 dilution

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 243 kDa

Observed band size: 250 kDa

false

• WB

Lab

Western blot - Anti-CAD antibody [EP710Y] - BSA and Azide free (AB231694)

This data was developed using ab40800, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CAD antibody [EP710Y] (<a href='/en-us/products/primary-antibodies/cad-antibody-ep710y-ab40800'>ab40800</a>) at 1/2000 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 243 kDa

Observed band size: 250 kDa

false