Recombinant Anti-PRMT4 antibody [EPR26711-36] (ab307091)

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lysates at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti-PRMT4 antibody EPR26711-36 ab307091 staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab307091 was shown to bind specifically to PRMT4. A band was observed at 63 kDa in wild-type HEK293T cell lysates with no signal observed at this size in PRMT4 knockout cell line (knockout cell lysate). To generate this image, wild-type and PRMT4 knockout HEK293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

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

In lane 1-4, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

In lanes 5-8, the lysates were stored at -80degC prior to Western Blotting. 

Bands above 250 kDa are aggregates of PRMT4.

Exposure time: 

Lane 1,2: 26 seconds

Lane 3,4: 48 seconds

Lane 5-8: 3 minutes

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized CARM1 KO HEK293T (CARM1 knockout human embryonic kidney epithelial cell)(ab266557) cells labeling PRMT4 with AB307091 at 1/500 dilution (1.026 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).

Confocal image showing nuclear and weak cytoplasmic staining in Parental HEK293T cell line, and no staining in CARM1 KO HEK293T cell line.

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 (2.5 ug/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 (2 ug/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (human breast adenocarcinoma epithelial cell) cells labeling PRMT4 with AB307091 at 1/500 dilution (1.026 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).

Confocal image showing nuclear and weak cytoplasmic staining in MCF7 cell line.

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 (2.5 ug/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 (2 ug/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling PRMT4 with AB307091 at 1/500 dilution (1.026 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).

Confocal image showing nuclear and weak cytoplasmic staining in NIH/3T3 cell line.

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 (2.5 ug/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 (2 ug/ml).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HEK-293T (human embryonic kidney epithelial cell, Right) / CARM1 knockout HEK-293T (Left) cells labeling PRMT4 with AB307091 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Positive staining on HEK-293T cells (ab255449), while no staining on CARM1 knockout HEK-293T cells (ab266557).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling PRMT4 with AB307091 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

PRMT4 was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell lysate 10 ug with AB307091 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using AB307091 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: 293T whole cell lysate 10 ug

Lane 2: AB307091 IP in 293T whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab307091 in 293T whole cell lysate

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

Exposure time: 41 seconds.

PRMT4 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug with AB307091 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using AB307091 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 ug

Lane 2: AB307091 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab307091 in NIH/3T3 whole cell lysate

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

Exposure time: 41 seconds.