Overview

  • Product name
    Anti-TopBP1 antibody - ChIP Grade
    See all TopBP1 primary antibodies
  • Description
    Rabbit polyclonal to TopBP1 - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, WB, IHC-P, ChIP, ICC/IF, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Human, African green monkey
    Predicted to work with: Chimpanzee, Gorilla, Orangutan
  • Immunogen

    Synthetic peptide (Human) - which represents a portion of human Topoisomerase (DNA) II Binding Protein I encoded within exon 28.

  • Positive control
    • HeLa cells and HCT116

Properties

Applications

Our Abpromise guarantee covers the use of ab2402 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP Use a concentration of 0.5 - 2.5 µg/ml. May cross-react with other proteins such as CHK1 or possibly claspin.
WB 1/1000 - 1/10000. Detects a band of approximately 200 kDa (predicted molecular weight: 173.3 kDa).
IHC-P Use a concentration of 4 µg/ml.
ChIP Use at an assay dependent concentration. PubMed: 17913746
ICC/IF Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.

Target

  • Function
    Required for DNA replication. Plays a role in the rescue of stalled replication forks and checkpoint control. Binds double-stranded DNA breaks and nicks as well as single-stranded DNA. Recruits the SWI/SNF chromatin remodeling complex to E2F1-responsive promoters. Down-regulates E2F1 activity and inhibits E2F1-dependent apoptosis during G1/S transition and after DNA damage. Induces a large increase in the kinase activity of ATR.
  • Tissue specificity
    Highly expressed in heart, brain, placenta, lung and kidney.
  • Sequence similarities
    Contains 8 BRCT domains.
  • Post-translational
    modifications
    Phosphorylated on serine and threonine residues in response to X-ray irradiation.
    Ubiquitinated and degraded by the proteasome. X-ray irradiation reduces ubiquitination.
  • Cellular localization
    Nucleus. Cytoplasm, cytoskeleton, microtubule organizing center, centrosome. Cytoplasm, cytoskeleton, spindle pole. Chromosome. Detected on unpaired autosomes in meiotic prophase cells. Detected on X and Y chromosomes during later stages of prophase. Colocalizes with ATR and H2AFX at unsynapsed chromosome cores during prophase (By similarity). Has a uniform nuclear distribution during G phase. Colocalizes with BRCA1 at stalled replication forks during S phase. In mitotic cells it colocalizes with BRCA1 at spindle poles and centrosomes during metaphase and anaphase. Detected in discrete foci together with PML and numerous DNA repair enzymes after DNA damage by alkylating agents, UV or gamma irradiation. Localizes to sites of DNA damage in a H2AX- independent manner.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA topoisomerase 2 binding protein 1 antibody
    • DNA topoisomerase 2-binding protein 1 antibody
    • DNA topoisomerase II binding protein 1 antibody
    • DNA topoisomerase II binding protein antibody
    • DNA topoisomerase II-beta-binding protein 1 antibody
    • DNA topoisomerase II-binding protein 1 antibody
    • DNA topoisomerase IIbeta binding protein 1 antibody
    • Hypothetical protein KIAA0259 [Fragment] antibody
    • KIAA0259 antibody
    • TOP2BP1 antibody
    • TOPB1_HUMAN antibody
    • TOPBP 1 antibody
    • TopBP1 antibody
    • Topoisomerase (DNA) II binding protein 1 antibody
    • Topoisomerase II binding protein 1 antibody
    see all

Images

  • Lane 1: WB Cell Lysate (5% of input IP)
    Lane 2: IP control (normal rabbit IgG)
    Lane 3: IP/WB

    Conditions:
    IP: Rabbit polyclonal to TopBP1 (1 µg)
    WB: Rabbit polyclonal to TopBP1 (1/2000 dilution)
    Positive control: HeLa Cells

    Lane 1: WB Cell Lysate (5% of input IP)
    Lane 2: IP control (normal rabbit IgG)
    Lane 3: IP/WB

    Conditions:
    IP: Rabbit polyclonal to TopBP1 (1 µg)
    WB: Rabbit polyclonal to TopBP1 (1/2000 dilution)
    Positive control: HeLa Cells

  • ab2402 (4µg/ml) staining TOPBP1 in human duodenum using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining in the epithelium.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Immunofluorescence analysis of African Green Monkey CV-1 (kidney epithelial) cells, staining TopBP1 with ab2402.

    Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 and blocked with 3% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/500 in 1% BSA in PBS) for 16 hours at 4°C. An AlexaFluor®488-conjugated donkey anti-rabbit polyclonal IgG (1/1000) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
See all 24 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

Application
Immunoprecipitation
Immuno-precipitation step
Protein A/G
Sample
Human Cell lysate - whole cell (HeLa cells)
Specification
HeLa cells
Total protein in input
30 µg

Abcam user community

Verified customer

Submitted Jan 07 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 45 minute(s) · Concentration: 2% · Temperature: RT°C
Sample
Human Cell (HeLa cells)
Specification
HeLa cells
Permeabilization
Yes - 0.2% Triton-X100
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Nov 24 2014

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (4-16% gradient Tris-Gly gel)
Sample
Mouse Cell lysate - whole cell (MEFs)
Specification
MEFs
Blocking step
LI-COR­ Odyssey­ Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Aug 01 2014

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (4-16% gradient Tris-Gly gel)
Sample
Human Cell lysate - whole cell (HeLa cells)
Specification
HeLa cells
Blocking step
LI-COR­ Odyssey­ Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Aug 01 2014

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing (3-8%)
Sample
Human Cell lysate - whole cell (HCT116)
Specification
HCT116
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Dec 17 2013

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Xenopus laevis Cell (Nuclei generated in oocyte extract)
Specification
Nuclei generated in oocyte extract
Fixative
Formaldehyde
Permeabilization
Yes - PBS-0.1% NP40
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 23°C

Dr. Andrew Lane

Verified customer

Submitted Apr 11 2013

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Xenopus laevis Cell lysate - whole cell (Xenopus egg extract)
Loading amount
40 µg
Specification
Xenopus egg extract
Gel Running Conditions
Reduced Denaturing (8%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Dr. Andrew Lane

Verified customer

Submitted Apr 11 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
African Green Monkey Cell (CV-1, Epithel Kidney)
Specification
CV-1, Epithel Kidney
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jan 28 2013

Question

I complete the IP questionnaire. I want to know if you are able to identify who is the band under TopBP1 in the input and in the IP. I attach apicture of IP/wester blot. I hope that you can help me

thank you

Abcam IP Questionnaire
1) Abcam product code ab2402

2) Abcam order reference number or product batch number #1109783

3) Description of the problem we identify an interaction between our protein of study and TopBP1 but there is another interaction more specific then TopBP1 recognized by TopBP1 antibody also in input of hela cell lines extract. The IP to TopBP1 is ok but I want to know what is the other band. The MW of Claspin is 150Kda but it migrates at 250Kda because of its iso-electric point and Chk1 is 55Kda

4) Sample preparation:

Type of sample whole Hela cell lysates

Lysis buffer: Hepes-NaOH 50mM ph 7,5 , NaCl 150mM, 1% glicerol, 1% Triton X100, MgCl2 1,5mM, EGTA 5mM

Protease inhibitors:protease inhibitor cocktail 78410 as recipe

Phosphatase inhibitors phosphatase inhibitor cocktail 1861277 as recipe

Amount of Lysate IPed ug or number of cells 2mg of lysate for IP

Lysate precleared with matrix : yes

Positive control whole Hela cell lysates 50ug

Negative control No IP only matrix


5) IP antibody:

Amount/concentration or dilution used 2ug of antibody for IP

Conjugation:

Antibody noncovalently bound to matrix before incubation with lysate sample? NO

Antibody noncovalently bound to matrix after incubation with lysate sample? YES

Antibody covalently bound to matrix - please describe briefly? NO

We used to pre-clear the cell lysates for 1h at 40C on roacking with 30ul of beads then pellet it for 5’ at 2000rpm 40C, collect the supernatant and put it to contct with antibodies O.N. Next day we used 20ul of beads/IP to bound the immunocomplex

Antibody-lysate incubation time: Over night

Incubation temperature 40C on roacking

6) IP Matrix (e. beads):

Type of matrix Protein G sepharose 4 fast flow GE Healthcare 17-0618-01

Amount of matrix 20ul

7) Washing after antibody-lysate incubation:

Buffer: : Hepes-NaOH 50mM ph 7,5 , NaCl 150mM, 1% glicerol, 1% Triton X100, MgCl2 1,5mM, EGTA 5mM

Number of washes 5

8) IP analysis:

Verification method: e.g reprobe in western blotting NO

Describe verification method briefly:

We used ECL Plus Western Blotting Detection Reagents Amersham

OR if Western blotting reprobe performed please indicate the following:

Reducing agent Bme

Boiling for ≥5 min? yes

Protein loaded ug/lane or cells/lane 50ug for input

Percentage of gel 10%

Type of membrane Amersham Hybond ECL Nitrocellulose Membrane

Protein transfer verified: YES, Ponceau

Blocking agent and concentration 5% milk in TBS+1% Tween

Blocking time 1h

Blocking temperature RT

Primary antibody (If more than one was used, describe in “additional notes”) :TopBP1 ab2402

Concentration or dilution 1:1000

Diluent buffer 5% milk in TBS+1% Tween

Incubation time Oven night

Incubation temperature: 40C

Secondary antibody:

Species: Goat

Reacts against: Rabbit

Concentration or dilution 1:3000

Diluent buffer 5% milk in TBS+1% Tween

Incubation time 45’

Incubation temperature: RT

Fluorochrome or enzyme conjugate: horseradish peroxidase conjugated

Washing after primary and secondary antibodies:

Buffer TBS+1% Tween

Number of washes 5

Detection method

Was the method successfully used to detect protein from non-IPed samples? Yes this methods detect the band of TopBP1 and another with smaller MW

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

Read More
Answer

Thank you for your enquiry regardingab2402 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

We regret to inform you that we do not know the identity of the band in question. We only know the identity for the target band TopBP1.

Does the detection system work fine? Have you used it successfully with another primary antibody? Have you run a no primary - only secondary antibody - control to see if any of the non-specific bands are due to the secondary or not? If you have not done yet, I would advise you to check it.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to better understand the problem, I would like to send you a questionnaire with some questions about the protocol used, as well as the order details. All this information is crucial to help us understand the possible causes of the problem. Any images are highly appreciated.

Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. If no suggestions can be made to the protocol, and the antibody was purchased within the last 6 months, it is covered by our Abpromise guarantee, and therefore I can offer you a free of charge replacement or a credit note for it.

I look forward to receiving your reply. Please do not hesitate to contact us if you need any more advice or information.

Read More

1-10 of 17 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up