Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling ATG12 with ab303488 at 1/100 (4.91 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast carcinoma. The section was incubated with ab303488 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded A Wild-type HAP1 (H tissue labeling ATG12 with ab303488 at 1/2000 (0.246 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) wild-type HAP1 cell pellet, no staining on (B) ATG12 knockout HAP1 cell pellet. The section was incubated with ab303488 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation.

ATG12 was immunoprecipitated from 0.35 mg Hela (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug with ab303488 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab303488 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab303488 IP in Hela whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab303488 in Hela whole cell lysate

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

Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-ATG12 antibody [EPR27485-89] (<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a>) at 1/1000 dilution

Lane 1:

Hela (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2:

<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a> IP in Hela whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

Exposure time: 180s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling ATG12 with ab303488 at 1/100 (4.91 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse testis. The section was incubated with ab303488 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling ATG12 with ab303488 at 1/100 (4.91 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat testis. The section was incubated with ab303488 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse breast carcinoma tissue labeling ATG12 with ab303488 at 1/100 (4.91 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse breast carcinoma. The section was incubated with ab303488 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling ATG12 with ab303488 at 1/50 (9.82 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image of NIH/3T3 cells treated with Chloroquine (200µM) for 2 hours. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. ATG12 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10ug with ab303488 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab303488 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10ug Lane 2 : ab303488 IP in NIH/3T3 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab303488 in NIH/3T3 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-ATG12 antibody [EPR27485-89] (<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10ug

Lane 2:

<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a> IP in NIH/3T3 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, ab303488 was shown to bind specifically to Atg5 bound Atg12 proteins. A band was observed at 52kDa in wild-type HAP1 cell lysate with no signal observed at this size in Atg12 knockout cell line. Exposure time : 180 seconds

All lanes:

Western blot - Anti-ATG12 antibody [EPR27485-89] (<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell line), whole cell lysate at 20 µg

Lane 2:

Atg12 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HCT116 (human colorectal carcinoma 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

Observed band size: 52 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : Lane 1-2 : 125 seconds Lane 3-4 : 26 seconds

All lanes:

Western blot - Anti-ATG12 antibody [EPR27485-89] (<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate ####20000#dilution#Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a> at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma 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

Observed band size: 52 kDa

false

• WB

Supplier Data

Western blot - Anti-ATG12 antibody [EPR27485-89] - BSA and Azide free (AB303489)

This data was developed using ab303488, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : 26 seconds

All lanes:

Western blot - Anti-ATG12 antibody [EPR27485-89] (<a href='/en-us/products/primary-antibodies/atg12-antibody-epr27485-89-ab303488'>ab303488</a>) at 1/1000 dilution

Lane 1:

Mouse pancreas tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lanes 3 - 4:

Rat brain tissue 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

Observed band size: 52 kDa

false

Exposure time: 26s