Anti-ATG4B antibody [EPR16572] - BSA and Azide free

• IP

Supplier Data

Immunoprecipitation - Anti-ATG4B antibody [EPR16572] - BSA and Azide free (AB251285)

This data was developed using ab199537, the same antibody clone in a different buffer formulation.

ATG4B was immunoprecipitated from 1mg of Ramos (Human Burkitt's lymphoma cell line) whole cell lysate with ab199537 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab199537 at 1/500 dilution (Panel A) or ab154843 at 1/500 dilution (Panel B). Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : Ramos whole cell lysate 10ug (Input). Lane 2 : ab199537 IP in Ramos whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab199537 in Ramos whole cell lysate.

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

Exposure time : Panel A and B 30 seconds.

All lanes:

Immunoprecipitation - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>)

Predicted band size: 44 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-ATG4B antibody [EPR16572] - BSA and Azide free (AB251285)

This data was developed using ab199537, the same antibody clone in a different buffer formulation.

ATG4B was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate with ab199537 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab199537 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : Jurkat whole cell lysate 10ug (Input). Lane 2 : ab199537 IP in Jurkat whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab199537 in Jurkat whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>)

Predicted band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-ATG4B antibody [EPR16572] - BSA and Azide free (AB251285)

This data was developed using ab199537, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>) at 1/1000 dilution

Lane 1:

293T (Human epithelial cells from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Ramos (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa

Observed band size: 44 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-ATG4B antibody [EPR16572] - BSA and Azide free (AB251285)

This data was developed using the same antibody clone in a different buffer formulation (ab199537).

Lanes 1 - 2 : Merged signal (red and green). Green - ab199537 observed at 47 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab199537 was shown to react with ATG4B in wild-type HeLa cells in Western blot with loss of signal observed in ATG4B knockout cell line ab265814 (ATG4B knockout cell lysate ab257364). Wild-type and ATG4B knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab199537 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATG4B knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATG4B knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-atg4b-knockout-hela-cell-line-ab265814'>ab265814</a>)

Predicted band size: 44 kDa

Observed band size: 47 kDa

false

• WB

Lab

Western blot - Anti-ATG4B antibody [EPR16572] - BSA and Azide free (AB251285)

This data was developed using ab199537, the same antibody clone in a different buffer formulation.

Lane 1 : Wild-type HAP1 cell lysate (20 μg)

Lane 2 : ATG4B knockout HAP1 cell lysate (20 μg)

Lane 3 : HeLa cell lysate (20 μg)

Lane 4 : Jurkat cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab199537 observed at 44 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab199537 was shown to specifically react with ATG4B when ATG4B knockout samples were used. Wild-type and ATG4B knockout samples were subjected to SDS-PAGE. ab199537 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>)

Predicted band size: 44 kDa

Observed band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-ATG4B antibody [EPR16572] - BSA and Azide free (AB251285)

This data was developed using ab199537, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ATG4B antibody [EPR16572] (<a href='/en-us/products/primary-antibodies/atg4b-antibody-epr16572-ab199537'>ab199537</a>) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa

Observed band size: 44 kDa

false

Exposure time: 1min