PRMT5 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate 10 ug with ab109451 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109451 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10 ug

Lane 2: ab109451 IP in Mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109451 in mouse brain tissue lysate

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

Exposure time: 100 seconds

All lanes : Anti-PRMT5 antibody [EPR5772] (ab109451) at 1/50000 dilution (purified)

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 73 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

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

Expression and localization of candidate epigenetic reprogramming factors in human embryos.

Human blastocysts were incubated with primary antibodies for (Panel B) PRMT5 (green), the nuclear DNA stained with DAPI (blue) and visualized by multi-channel confocal microscopy. Note that the expression of PRMT5 primarily localized to the ICM of human blastocysts (indicated by white arrows).

Anti-PRMT5 antibody [EPR5772] (ab109451) at 1/10000 dilution (purified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 73 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

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

All lanes : Anti-PRMT5 antibody [EPR5772] (ab109451) at 1/10000 dilution (purified)

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 73 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

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

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling PRMT5 (green) with purified ab109451 at 1/50.

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: Secondary antibody Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/400).

ICC/IF image of ab109451 (unpurified) stained HepG2 (Human liver hepatocellular carcinoma cell line) cells.

The cells were fixed with 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab109451, 1/100 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight^® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling PRMT5 with purified ab109451 at 1/100.

A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody.

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human infiltrating duct carcinoma of breast tissue sections labeling PRMT5 with purified ab109451 at 1/100.

A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody.

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa (Human liver hepatocellular carcinoma cell line) cells stained with ab109451 (unpurified, red line).

The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109451, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monclonal) (1 µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

Equilibrium disassociation constant (K_D)

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