Recombinant Anti-ATG12 antibody [EPR27485-89] (ab303488)

All lanes : Anti-ATG12 antibody [EPR27485-89] (ab303488) at 1/1000 dilution

Lane 1 : Mouse pancreas tissue lysate
Lane 2 : Mouse testis tissue lysate
Lane 3 : Rat brain tissue lysate
Lane 4 : Rat testis tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 15 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?


Exposure time: 26 seconds

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes : Anti-ATG12 antibody [EPR27485-89] (ab303488) at 1/1000 dilution

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 15 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

Exposure time:

Lane 1-2: 125 seconds
Lane 3-4: 26 seconds

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes : Anti-ATG12 antibody [EPR27485-89] (ab303488) at 1/1000 dilution

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell line), whole cell lysate
Lane 2 : Atg12 knockout HAP1 whole cell lysate
Lane 3 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 15 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

Blocking / 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.

ATG12 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10µg 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.

ATG12 was immunoprecipitated from 0.35 mg Hela (human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg 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.

Immunohistochemical analysis of paraffin-embedded sections of (A) Wild-type HAP1 (Human chronic myelogenous leukemia near-haploid cell) cell pellet and (B) ATG12 knockout HAP1 cell pellet. ab303488 used at 1/2000 (0.246 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit) was used. 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 Leica DS9800 (Bond™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded human breast carcino tissue labeling ATG12 with ab303488 at 1/100 (4.91 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit. 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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling ATG12 with ab303488 at 1/100 (4.91 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit. 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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse breast carcinoma tissue labeling ATG12 with ab303488 at 1/100 (4.91 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit. 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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling ATG12 with ab303488 at 1/100 (4.91 µg/ml) followed by a ready to use Leica DS9800 (Bond^™ Polymer Refine Detection) kit. 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 Leica DS9800 (Bond^™ Polymer Refine Detection) kit.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling ATG12 with ab303488 at 1/50 (9.82 µg/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 2µg/mL) (Green). Confocal image of NIH/3T3 cells treated with Chloroquine (200 µM) for 2 hours. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5µg/mL) (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 dilution (2µg/mL).