All lanes : Anti-GST3 / GST pi antibody [EPR20554] (ab241331) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Human heart tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/100000 dilution

Predicted band size: 23 kDa
Observed band size: 23 kDa


Exposure time: 52 seconds

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

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling GST3 / GST pi with ab241331 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on tubules of human kidney (PMID: 1977319). Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling GST3 / GST pi with ab241331 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on subset immune cells of human tonsil. (PMID: 18604726). Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling GST3 / GST pi with ab241331 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on duct epithelium of human pancreas. (PMID: 1977319). Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (human T cell leukemia T lymphocyte) cells labeling GST3 GST pi (Green) with ab241331 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing cytoplasmic staining in Jurkat cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized wild type and GST pi KO HAP1 (GST pi knockout human chronic myelogenous leukemia near-haploid cell line) cells labeling GST3 / GST pi (Green) with ab241331 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing cytoplasmic staining in parental HAP1 cell line and no staining in GST pi KO HAP1 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized GST pi KO HAP1 (human chronic myelogenous leukemia near-haploid cell line, Left) / Wild-type HAP1 (Right) cell line labeling GST3 / GST pi with ab241331 at 1/300 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-GST3 / GST pi antibody [EPR20554] (ab241331) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : GST3 / GST pi knockout HAP1 cell lysate
Lane 3 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 5 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 23 kDa
Observed band size: 23 kDa


Exposure time: 15 seconds

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

ab241331 was shown to specifically react with GST3 / GST pi in wild-type HAP1 cells as signal was lost in GST3 / GST pi knockout cells. Wild-type and GST3 / GST pi knockout samples were subjected to SDS-PAGE. ab241331 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.