Anti-Peregrin/BRPF1 antibody [EPR24069-57]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling Peregrin/BRPF1 with ab259840 at 1/50 (11.34 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cells is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma cell) cells labelling Peregrin/BRPF1 with ab259840 at 1/500 dilution (0.1μg) (Red) 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 4T1 (mouse mammary gland carcinoma epithelial cell) cells labelling Peregrin/BRPF1 with ab259840 at 1/500 dilution (0.1μg) (Red) 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling Peregrin/BRPF1 with ab259840 at 1/50 (11.34 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cells is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (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.

• WB

Lab

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 7906940).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (ab259840) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 137 kDa

Observed band size: 150-160 kDa

false

• WB

Lab

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

False colour image of Western blot : Anti-Peregrin/BRPF1 antibody [EPR24069-57] staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab259840 was shown to bind specifically to Peregrin/BRPF1. A band was observed at 120 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in BRPF1 knockout cell line ab266549 (knockout cell lysate ab258795). To generate this image, wild-type and BRPF1 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) 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.

Lanes 1, 2, 3, 5 and 6:

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (ab259840) at 1/500 dilution

Lane 4:

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (ab259840)

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lanes 2 - 3:

BRPF1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human BRPF1 (Peregrin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-brpf1-peregrin-knockout-hek-293t-cell-line-ab266549'>ab266549</a>)

Lane 4:

Empty

Lane 5:

PC-3 cell lysate at 20 µg

Lane 6:

HepG2 cell lysate at 20 µg

Predicted band size: 137 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (AB259840)

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

Exposure time : 8 seconds

All lanes:

Western blot - Anti-Peregrin/BRPF1 antibody [EPR24069-57] (ab259840) at 1/5000 dilution

Lane 1:

His-tagged human BRPF1 recombinant protein at 0.01 µg

Lane 2:

His-tagged human BRPF3 recombinant protein at 0.2 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 137 kDa

false