Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using ab175188, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling 53BP1 with ab175188 at a 1/50 dilution. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using ab175188, the same antibody clone in a different buffer formulation.

Immunofluorescence analysis of HepG2 cells labeling 53BP1 with ab175188 at a 1/100 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using ab175188, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human prostate hyperplasia tissue labeling 53BP1 with ab175188 at a 1/50 dilution. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175188).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling 53BP1 with ab175188 at 1/500 (0.976 µg/ml) dilution followed by a ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at a 1/1000 (2 µg) dilution (Green).

Confocal image showing nuclear staining in NIH/3T3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). 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 (2.5µg/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at a 1/1000 (2 µg) dilution.

• WB

Lab

Western blot - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175188).

Lanes 1 - 3 : Merged signal (red and green). Green - ab175188 observed at 213 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab175188 was shown to recognize 53BP1 in wild-type HAP1 cells as signal was lost at the expected MW in TP53BP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and TP53BP1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab175188 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/50000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (ab249845) at 1/50000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

TP53BP1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 214 kDa

false

• WB

Lab

Western blot - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using ab175188, the same antibody clone in a different buffer formulation.

Lanes 1 - 3 : Merged signal (red and green). Green - ab175188 observed at 213 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab175188 was shown to recognize 53BP1 in wild-type HAP1 cells as signal was lost at the expected MW in TP53BP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and TP53BP1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab175188 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/50000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-53BP1 antibody [EPR2173(2)] (<a href='/en-us/products/primary-antibodies/53bp1-antibody-epr21732-ab175188'>ab175188</a>) at 1/50000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

TP53BP1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Predicted band size: 214 kDa

false

• WB

Supplier Data

Western blot - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using ab175188, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-53BP1 antibody [EPR2173(2)] (<a href='/en-us/products/primary-antibodies/53bp1-antibody-epr21732-ab175188'>ab175188</a>) at 1/50000 dilution

Lane 1:

Human fetal heart tissue lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

Human fetal brain tissue lysate at 10 µg

Predicted band size: 214 kDa

false

• WB

Lab

Western blot - Anti-53BP1 antibody [EPR2173(2)] - BSA and Azide free (AB249845)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175188).

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

ab129002 was used as a Vinculin loading control at a 1/10000 dilution.

In Western blot, ab175188 was shown to bind specifically to Dnmt1. Target of interest was observed mainly at 270 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in Dnmt1 knockout cell line (lane 2) .

All lanes:

Western blot - Anti-53BP1 antibody [EPR2173(2)] (<a href='/en-us/products/primary-antibodies/53bp1-antibody-epr21732-ab175188'>ab175188</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Dnmt1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 5:

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

Lane 6:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) 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

Observed band size: 270 kDa

false

Exposure time: 70s