Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling KDM3A / JHDM2A with ab243641 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat testis (PMID: 17943087). The section was incubated with ab243641 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling KDM3A / JHDM2A with ab243641 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line treatment with DFO (1mM) for 24h. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa cells treated with 1mM DFO for 24h (Red) / Untreated control (Green)  labelling KDM3A / JHDM2A with ab243641 at 1/500 dilution (0.1ug) (Red) / (Green) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

KDM3A / JHDM2A was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate with ab243641 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab243641 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate 10 ug

Lane 2: ab243641 IP in Ramos whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab243641 in Ramos whole cell lysate

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

Exposure time: 3 minutes.

 

All lanes : Anti-KDM3A / JHDM2A antibody [EPR18875-106] (ab243641) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2 : HeLa treated with 1mM Deferoxamine mesylate (DFO) for 24 hours, whole cell lysate

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 147 kDa
Observed band size: 147 kDa

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

Fresh lysates were used in this WB.

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling KDM3A / JHDM2A with ab243641 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse testis (PMID: 17943087). The section was incubated with ab243641 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Anti-KDM3A / JHDM2A antibody [EPR18875-106] (ab243641) at 1/1000 dilution + NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 147 kDa
Observed band size: 147,60,90 kDa why is the actual band size different from the predicted?

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

This blot was developed using a higher sensitivity ECL substrate.

Fresh lysates were used in this WB.

The bands at 90, 60KD are the degragated fragments of KDM3A which is consistent with the literature (PMID: 31629659, 26728187).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling KDM3A / JHDM2A with ab243641 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human testis (PMID: 25071150). The section was incubated with ab243641 for 30 mins at room temperature.T he immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Anti-KDM3A / JHDM2A antibody [EPR18875-106] (ab243641) at 1/1000 dilution + Ramos (human Burkitts lymphoma B lymphocyte), whole cell lysate at 30 µg

Secondary
VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/5000 dilution

Predicted band size: 147 kDa
Observed band size: 147 kDa

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

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 3 minutes.

Anti-KDM3A / JHDM2A antibody [EPR18875-106] (ab243641) at 1/1000 dilution + C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 147 kDa
Observed band size: 147,60 kDa why is the actual band size different from the predicted?

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

This blot was developed using a higher sensitivity ECL substrate.

Fresh lysates were used in this WB.

The band at 60KD is the degragated fragment of KDM3A which is consistent with the literature(PMID: 31629659, 26728187). 

Exposure time: 3 minutes.