What is the recommended pH of sodium citrate for the 53bp1 antibody (NB100-304)?

I have checked our notes for use of this antibody in IHC, and they say: 1:500 to 1:2,000. Epitope retrieval with citrate buffer pH6.0 is recommended for FFPE tissue sections.

Is possible to know the exact sequence of the epitope used?

Unfortunately I am unable to disclose the sequence of this antibody due to proprietary restrictions. At the site recognized by NB100-304, the reference sequences are too divergent to expect NB100-304 to bind to either Bovine or Little Brown Bat. Regarding Mouse Homology, 30/34 sites of the immunogen are homologous Human - 100% homologous. If you would be interested in testing these novel species, please take a look at our Innovator's Reward program.

53BP1 Antibody - BSA Free

Name: 53BP1 Antibody - BSA Free
Brand: Novus Biologicals
SKU: NB100-304
Rating: 5 (14 reviews)

Novus Biologicals, part of Bio-Techne | Catalog # NB100-304

(14)

Citations (774)

Product Datasheet / COA / SDS / Protocols & Manuals

View Available Conjugates

Conjugate

Catalog #

Alexa Fluor 350

NB100-304AF350

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Alexa Fluor 405

NB100-304AF405

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Alexa Fluor 488

NB100-304AF488

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Alexa Fluor 532

NB100-304AF532

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Alexa Fluor 594

NB100-304AF594

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Alexa Fluor 647

NB100-304AF647

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Alexa Fluor 700

NB100-304AF700

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Alexa Fluor 750

NB100-304AF750

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Allophycocyanin

NB100-304APC

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Allophycocyanin/Cy7

NB100-304APCCY7

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Biotin

NB100-304B

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DyLight 350

NB100-304UV

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DyLight 405

NB100-304V

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DyLight 488

NB100-304G

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DyLight 550

NB100-304R

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DyLight 594

NB100-304DL594

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DyLight 650

NB100-304C

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DyLight 680

NB100-304FR

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DyLight 755

NB100-304IR

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FITC

NB100-304F

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HRP

NB100-304H

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Janelia Fluor 549

NB100-304JF549

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Janelia Fluor 646

NB100-304JF646

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mFluor Violet 450 SE

NB100-304MFV450

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mFluor Violet 500 SE

NB100-304MFV500

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mFluor Violet 610 SE

NB100-304MFV610

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NB100-304PE

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PE/Atto594

NB100-304PEATT594

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PE/Cy5.5

NB100-304PECY55

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PE/Cy7

NB100-304PECY7

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PerCP

NB100-304PCP

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Product Specifications
Scientific Data
Applications
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Validated by

Independent Antibodies, Tagged Protein Expression

Species Reactivity

Validated:

Human, Mouse, Rat, Bat, Bovine, Canine, Fish, Goat, Primate

Cited:

Human, Mouse

Predicted:

Chimpanzee (96%), Feline (90%), Orangutan (96%), Porcine (90%), Rabbit (90%), Sheep (90%). Backed by our 100% Guarantee.

Applications

Validated:

Chromatin Immunoprecipitation, Chromatin Immunoprecipitation (ChIP), Flow (Intracellular), Flow Cytometry, Immunoblotting, Immunocytochemistry/Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, In-situ Hybridization, Knockdown Validated, Knockout Validated, Western Blot

Cited:

Immunocytochemistry/ Immunofluorescence, Proximity Ligation Assay, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1 mg/ml

View all 53BP1 Antibodies »

Product Specifications

Immunogen

Partial synthetic peptide made to an internal portion of human 53BP1 (between amino acids 350-400) [NP_005648.1].

Reactivity Notes

Human reactivity reported in scientific literature (PMID:32877678). Human and mouse reactivity cited in numerous publications. Primate reactivity reported in scientific literature (PMID: 18389475). Fish reactivity reported in scientific literature (PMID: 25516420). Bat, canine, and bovine reactivity reported in scientific literature (PMID: 27573809). Predicted cross-reactivity based on sequence identity: Chimpanzee (96%), Gorilla (96%), Orangutan (96%), Gibbon (94%), Marmoset (92%), Feline (90%), Porcine (90%), Rabbit (90%), Sheep (90%).

Marker

DNA Double Strand Break Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

214 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for 53BP1 Antibody - BSA Free

Immunohistochemical Detection of 53BP1 in Frozen Mouse Ganglion Cells

Irradiated cochlear spiral ganglion cells, mouse, frozen section of fixed material. Image from verified customer review.

Knockout Validation of 53BP1 Antibody in HeLa and 53BP1 Knockout HeLa Cells in Immunocytochemistry/Immunofluorescence

53BP1 was detected in immersion fixed HeLa cells (left) but was not detected in 53BP1 knockout Hela cells (right) using Rabbit Anti-human 53BP1 polyclonal antibody (Catalog #NB100-304) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to nuclei.

Western Blotting of 53BP1 in Multiple Cell Lines

Total protein from HeLa, A431, Neuro2A, and PC12 was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1.0 ug/mL in 5% block buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. The observed molecular weight for these samples are ~250 kDa and the theoretical molecular weight is 214 kDa.

Immunohistochemical Analysis of 53BP1 in Paraffin Embedded Breast Tumors

Human breast tumors stained with 53BP1 antibody. Image from verified customer review.

53BP1 Staining in Multiple Tissues

Detection of Human and Mouse 53BP1 by IHC. Sample: FFPE sections of human ovarian carcinoma (left) and mouse teratoma (right). Antibody: 53BP1 Antibody (Catalog #NB100-304) used at a dilution of 1:1000 (1ug/mL). Detection: DAB.

Immunocytochemistry/Immunofluorescence Staining of 53BP1 in Multiple Cell Lines

Human medulloblastoma (DAOY) and mouse astrocyte (C8D1A) cell lines were exposed for 48 hours to DMSO or 1ug/mL of the DNA damaging agent EPE. Cells were immunostained for 53BP1 (green). The nuclei were counterstained with DAPI (blue). Image from verified customer review.

Intracellular Staining of 53BP1 in RH-30 Cells in Flow Cytometry

An intracellular stain was performed on RH-30 cells with 53BP1 Antibody (Catalog #NB100-304AF647) (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.

Detection of 53BP1 in Immunoprecipitated HCC44 Cell Lysate

Immunoprecipitation analysis lysates from HCC44 cells in 1% NP40. Image from verified customer review.

Fluorescent Detection of 53BP1 in Irradiated A549 Cells

Effect of RK-33 on radiation-induced DNA damage. A Immunofluorescence images showing 53BP1 and gamma-H2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 uM RK-33, 12 h before radiation. Overlap of 53BP1 and gamma-H2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 um. Image collected and cropped by CiteAb from the following publication (http://embomolmed.embopress.org/cgi/doi/10.15252/emmm.201404368) licensed under a CC-BY licence.

Western Blot of 53BP1 in U2OS Cells Exposed to Ionizing Radiation

Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. U2OS/GFP-lamin A cells were pretreated with 10 um ATMi f for 1 h before exposure to IR (10 Gy, 1 h recovery). Cell extracts were then subjected to immunoprecipitation using GFP-Trap beads, and bound complexes were then analyzed by immunoblotting using 53BP1 and GFP antibodies. WCE represents 1% input. Image collected and cropped by CiteAb from the following publication (http://doi.wiley.com/10.1111/acel.12258), licensed under a CC-BY licence.

Immunocytochemistry/Immunofluorescence Staining of 53BP1 in HeLa Cells

HeLa cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with 53BP1 Antibody conjugated to DyLight 550 (NB100-304R) at 5 ug/ml for 1 hour at room temperature. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Intracellular Staining of 53BP1 in Ntera2 Cells in Flow Cytometry

An intracellular stain was performed on Ntera2 cells with 53BP1 Antibody NB100-304 (blue) and a matched isotype control NBP2-24891 (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (SA5-10033, Thermo Fisher).

Western Blot of 53BP1 in Multiple Cell Lines

Whole cell lysate from U2OS or 293T cells. Bands indicate an observed molecular weight of ~220 kDa and the theoretical molecular weight is 214 kDa.

Immunocytochemistry/Immunofluorescence of Irradiated and Non-Irradiated MEFs

Upper Panel: 53BP1 foci in proliferating MEFs. Lower Panel: 53BP1 foci in proliferating MEFs exposed to 10 Gy of IR.

Immunocytochemistry/Immunofluorescence Staining of 53BP1 in Embryonic Fibroblast Cells

Embryonic Fibroblast cells pre-extraction for 5 mins with CSK buffer. Fixed with 4% PFA and 75% Ethanol. Primary Antibody at 1:1000. Secondary Antibody at 1:1000. Image from verified customer review.

Probing of 53BP1 in Untreated and Irradiated Cells

Upper Panel: Control untreated cells. Lower Panel: Cells exposed to irradiation (10 Gy) and probed for 53BP1 foci. Cells were grown on coverslips, fixed with 4% paraformaldehyde, methanol permeabilized, blocked for 1 h, RT. Incubated with primary antibody (1:200) overnight, washed 3x with PBS, probed with tubulin (Alexa Fluor 594) antibody for 2 h, RT. Washed 3x with PBS, mounted on slides using prolong gold, imaged using Nikon confocal microscope (100x oil). Image from verified customer review.

Immunocytochemistry/Immunofluorescence Staining of 53BP1 in Transfected H1299 Cells

MUS81 inhibition in BRCA2-deficient cells causes accumulation of 53BP1 nuclear bodies and G1 arrest. H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs. Representative images of cells processed 72 h later for immunostaining with 53BP1 Antibody (green) and anti-cyclin A (red) antibodies. DNA was counterstained with DAPI. Scale bar, 10 um. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/doifinder/10.1038/ncomms15983), licensed under a CC-BY licence.

Immunocytochemistry/Immunofluorescence of 53BP1 in Control and shPot1a-1 Transduced LSK Cells

Pot1a prevents DDR in HSCs. Telomeric DDR in 8 week-old LSK cells upon Pot1a knockdown. Immunocytochemical staining of TRF1 (green). Foci co-stained with TRF1 and 53BP1 were identified as TIFs. Nuclei were stained with TOTO3 (blue). Scale bar, 2 um. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/s41467-017-00935-4), licensed under a CC-BY licence.

Immunofluorescence of 53BP1 in Transfected U2OS Cells

Lamin A/C-53BP1 interaction is regulated in a DNA damage-dependent manner. U2OS cells were transfected with siCTRL or siLMNA and subjected to laser micro-irradiation, fixed 15 min later and then processed for immunofluorescence with gamma-H2AX and 53BP1 antibodies. Scale bar, 10 um. Image collected and cropped by CiteAb from the following publication (http://doi.wiley.com/10.1111/acel.12258), licensed under a CC-BY licence.

Immunofluorescent Staining of 53BP1 in Ntera2 Cells

Ntera2 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-53BP1 Antibody NB100-304 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:1000 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Immunofluorescent Staining of 53BP1 in NIH3T3 Cells

NIH3T3 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-53BP1 Antibody NB100-304 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:1000 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Immunocytochemistry/Immunofluorescence in HeLa Cells Using Conjugated 53BP1 Antibody

HeLa cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with 53PB1 Antibody conjugated to DyLight 550 (NB100-304R) at 5 ug/ml for 1 hour at room temperature. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Immunohistochemical Staining of 53BP1 in Paraffin Embedded Colon Cancer

Staining of 53BP1 in human colon cancer using DAB with hematoxylin counterstain.

Flow Cytometry of A431 Cells Stained with Conjugated 53BP1 Antibody

An intracellular stain was performed on A431 cells with 53BP1 Antibody (Catalog #NB100-304AF647) (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.

Cells Stained with 53BP1 Antibody in Immunocytochemistry/Immunofluorescence

For 53BP1 staining, samples were incubated for 1 hour with 1:200 53BP1 primary antibody in PBS/BSA 1%, at room temperature. Green: 53BP Blue: DAPI. Image from verified customer review.

View 22 more images

Applications for 53BP1 Antibody - BSA Free

Application

Recommended Usage

Chromatin Immunoprecipitation

reported in scientific literature (PMID 24591601)

Flow Cytometry

1.5 ug/ml

Immunoblotting

reported in multiple pieces of scientific literature

Immunocytochemistry/Immunofluorescence

1:1000-1:5000

Immunohistochemistry

reported in scientific literature (PMID 24987917)

Immunohistochemistry-Frozen

reported by customer review

Immunohistochemistry-Paraffin

1:1000-1:5000. Use reported in scientific literature (PMID 27653664)

Immunoprecipitation

reported in scientific literature (PMID 25645366)

In-situ Hybridization

reported in scientific literature (PMID 34988401; 33122290)

Knockout Validated

reported in scientific literature (PMID 26601238)

Western Blot

1:5000-1:25000

Reviewed Applications

Read 14 reviews rated 4.6 using NB100-304 in the following applications:

Immunocytochemistry (8 Reviews)
Immunofluorescence (1 Review)
Immunohistochemistry (1 Review)
Immunohistochemistry-Frozen (1 Review)
Immunoprecipitation (1 Review)
Western Blot (2 Reviews)

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at -20 degrees C. Avoid freeze/thaw cycles.

Background: 53BP1

Tumor protein p53 binding protein 1 (P53-binding protein 1 or 53BP1) plays a critical role in tumor suppression and is a putative substrate of ATM kinase with a theoretical molecular weight of 214 kDa. Upon DNA damage, it is phosphorylated and relocalizes to the presumptive sites of damage, specifically, double-strand breaks. 53BP1 plays a key role in response to DNA damage, acts as a signaling checkpoint during mitosis, and enhances TP53-mediated transcriptional activation. Originally identified as p53's transcriptional enhancing partner, 53BP1 is known as a key substrate for ataxia telangiectasia mutated (ATM) signaling, whose function to generate gamma H2AX may be partially compensated by the activity of DNA-dependent kinase (DNA-PK). 53BP1 relocalizes to discrete foci overlapping with gamma phosphorylated histone H2AX; demarcating DNA double strand breaks (DSBs) sites following exposure to radiation (1). 53BP1 functions downstream of gamma H2AX-dependent proteins that collectively establish ionizing radiation induced foci at DSBs. 53BP1 is downstream of Mre11/Rad50/NBS1 (MRN complex), MDC1, RNF8, RNF168 and HERC2 which recruit 53BP1 to the DSB site, suggesting a role in DNA repair through genomic stability maintenance (2).

References

1.Henry, E., Souissi-Sahraoui, I., Deynoux, M., Lefevre, A., Barroca, V., Campalans, A., . . . Arcangeli, M. L. (2019). Human hematopoietic stem/progenitor cells display ROS-dependent long-term hematopoietic defects after exposure to low dose of ionizing radiations. Haematologica. doi:10.3324/haematol.2019.226936

2.Janoshazi, A. K., Horton, J. K., Zhao, M. L., Prasad, R., Scappini, E. L., Tucker, C. J., & Wilson, S. H. (2020). Shining light on the response to repair intermediates in DNA of living cells. DNA Repair (Amst), 85, 102749. doi:10.1016/j.dnarep.2019.102749

Long Name

p53 Binding Protein 1

Alternate Names

p202, TP53BP1

Gene Symbol

TP53BP1

Additional 53BP1 Products

Product Documents for 53BP1 Antibody - BSA Free

Product Datasheets

Download Product Datasheets

Protocols & Manuals

Download Protocol

Download ICC/IF Protocol

Download Western Blot Protocol

Download IHC-P Protocol

COA

SDS

Download SDS

Product Specific Notices for 53BP1 Antibody - BSA Free

Please note for Lot A5, the dilution recommendations have changed from our previous lot recommendations. You may be required to use the antibody at a higher dilution than previous lots to obtain similar results. Please contact us if you have any questions.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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