Anti-53BP1 antibody [EPR2173(2)]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2173(2)] (AB175188)

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)] (AB175188)

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)] (AB175188)

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)] (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

Supplier Data

Western blot - Anti-53BP1 antibody [EPR2173(2)] (AB175188)

All lanes:

Western blot - Anti-53BP1 antibody [EPR2173(2)] (ab175188) 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)] (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)] (ab175188) 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)] (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)] (ab175188) 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

• WB

CiteAb

Western blot - Anti-53BP1 antibody [EPR2173(2)] (AB175188)

53BP1 western blot using anti-53BP1 antibody [EPR2173(2)] ab175188. Publication image and figure legend from Han, J., Ruan, C., et al., 2017, Nat Commun, PubMed 29133916.

ab175188 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab175188 please see the product overview.

Artemis functions epistatically with 53BP1 and RIF1 to promote alt-NHEJ. a 53BP1 or RIF1 depletion reduces the frequency of alt-NHEJ in both wild-type and BRCA2-depleted cells. 53BP1-, RIF1- or PTIP-depleted U2OS alt-NHEJ-EGFP cells were transfected with indicated siRNAs and then electroporated with pCBASce plasmid. After 48 h, the percentage of GFP-positive cells was determined by FACS. Results represent mean ± SEM of three independent experiments. b Knockdown efficiency was confirmed by Western blot. The Asterisk indicates a nonspecific band. c Artemis functions epistatically with 53BP1 and RIF1 to promote alt-NHEJ. Wild-type or Artemis-deficient U2OS alt-NHEJ-EGFP cells infected with the indicated lentiviral shRNAs were transfected with control siRNA or siRNA against BRCA2. Twenty-four hours post transfection, cells were electroporated with pCBASce plasmid. After 48 h, the percentage of GFP-positive cells was determined by FACS. Results represent mean ± SEM of three independent experiments. d Loss of 53BP1 or RIF1 suppresses gross genomic instability in BRCA2-depleted cells. Wild-type, 53BP1-, PTIP-, or RIF1-deficient HeLa cells were transfected with indicated siRNAs, exposed to 10 Gy IR, and then allowed to recover for 2 or 24 h before being processed for immunofluorescence using antibodies against RPA2 and RAD51. Representative RPA2 and RAD51 foci and DAPI-stained nuclei are shown. e Quantification of RPA2/RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. f RIF1 is required for the retention of Artemis at laser-induced DSBs. 53BP1- or RIF1-depleted HeLa cells were transfected with indicated siRNAs. Forty-eight hours later, cells were transfected with GFP-Artemis and were then micro-irradiated and monitored by live cell imaging. Representative images taken at the indicated times after laser microirradiation are shown. g The fluorescence intensity at the microirradiated site was quantified. Data represent mean ± SEM from at least 20 cells in three independent experiments. Scale bars, 10 μm

false