Overview

  • Product name
  • Description
    Rabbit polyclonal to 53BP1
  • Host species
    Rabbit
  • Specificity
    Recognises 53BP1
  • Tested applications
    Suitable for: In situ hybridization, WB, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide (C terminal).

  • Positive control
    • HeLa whole cell lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab21083 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
In situ hybridization Use at an assay dependent concentration. PubMed: 23370393
WB 1/500 - 1/3000. Detects a band of approximately 250 kDa (predicted molecular weight: 220 kDa).
ICC/IF 1/200. Used at a dilution of 1/200 for 30 min incubation (see Abreview submitted by Kirk McManus).
IHC-P Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function
    May have a role in checkpoint signaling during mitosis. Enhances TP53-mediated transcriptional activation. Plays a role in the response to DNA damage.
  • Involvement in disease
    Note=A chromosomal aberration involving TP53BP1 is found in a form of myeloproliferative disorder chronic with eosinophilia. Translocation t(5;15)(q33;q22) with PDGFRB creating a TP53BP1-PDGFRB fusion protein.
  • Sequence similarities
    Contains 2 BRCT domains.
  • Post-translational
    modifications
    Asymmetrically dimethylated on Arg residues by PRMT1. Methylation is required for DNA binding.
    Phosphorylated at basal level in the absence of DNA damage. Hyper-phosphorylated in an ATM-dependent manner in response to DNA damage induced by ionizing radiation. Hyper-phosphorylated in an ATR-dependent manner in response to DNA damage induced by UV irradiation.
  • Cellular localization
    Nucleus. Chromosome > centromere > kinetochore. Associated with kinetochores. Both nuclear and cytoplasmic in some cells. Recruited to sites of DNA damage, such as double stand breaks. Methylation of histone H4 at 'Lys-20' is required for efficient localization to double strand breaks.
  • Information by UniProt
  • Database links
  • Alternative names
    • 53 BP1 antibody
    • 53BP1 antibody
    • FLJ41424 antibody
    • MGC138366 antibody
    • p202 antibody
    • p53 binding protein 1 antibody
    • p53 BP1 antibody
    • p53-binding protein 1 antibody
    • p53BP1 antibody
    • TP53 BP1 antibody
    • TP53B_HUMAN antibody
    • Tp53bp1 antibody
    • TRP53 BP1 antibody
    • Tumor protein 53 binding protein 1 antibody
    • Tumor protein p53 binding protein 1 antibody
    • Tumor suppressor p53 binding protein 1 antibody
    • Tumor suppressor p53-binding protein 1 antibody
    see all

Images

  • ICC/IF analysis using ab21083 at 1/1000 dilution labeling 53BP1 in HeLa cells fixed in 4% paraformaldehyde at RT for 15 minutes. ab21083 detects 53BP1 protein at the nucleus (green).
    Red: alpha Tubulin, a cytoskeleton marker.

  • All lanes : Anti-53BP1 antibody (ab21083) at 1/500 dilution

    Lane 1 : 53BP1 shRNA non-transfected HeLa whole cell extracts
    Lane 2 : 53BP1 shRNA transfected HeLa whole cell extracts

    Lysates/proteins at 50 µg per lane.

    Predicted band size: 220 kDa

  • ab21083 (2µg/ml) staining 53BP1 in human Brain: Cerebellum using an automated system (DAKO Autostainer Plus). Using this protocol there is ubiquitous nuclear staining throughout.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer 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.
  • All lanes : Anti-53BP1 antibody (ab21083) at 1/2000 dilution

    Lane 1 : 293T whole cell lysate/extract
    Lane 2 : A431 whole cell lysate/extract
    Lane 3 : HeLa whole cell lysate/extract
    Lane 4 : HepG2 whole cell lysate/extract
    Lane 5 : A375 whole cell lysate/extract

    Lysates/proteins at 30 µg per lane.

    Predicted band size: 220 kDa



    5 % SDS-PAGE

  • All lanes : Anti-53BP1 antibody (ab21083) at 1/500 dilution

    Lanes 1 & 3 : Neuro2A whole cell extracts
    Lane 2 : C8D30 whole cell extracts

    Lysates/proteins at 30 µg per lane.

    Predicted band size: 220 kDa

References

This product has been referenced in:
  • Komseli ES  et al. A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence. BMC Genomics 19:37 (2018). Read more (PubMed: 29321003) »
  • Toro C  et al. A recurrent de novo missense mutation in UBTF causes developmental neuroregression. Hum Mol Genet 27:691-705 (2018). Read more (PubMed: 29300972) »
See all 45 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (head and neck cancer)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (8)
Loading amount
15 µg
Treatment
drug 24h
Specification
head and neck cancer
Blocking step
BSA as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 06 2015

Answer

Thank you very much for your reply.

That is no problem to arrange. I have organised a new order for you for a new vial of ab21083. This is on the order number xxxx(Purchase order numberxxxxx). Please let me know if you have any problems receiving this.

I hope this one performs better for you. However, if you do continue to have problems please do let me know.

Until then, I wish you all the best with your research.

Read More

Question

Dear xxxx,

Thanks for your email. I’ve copied the questionnaire below and also attached the images for your reference.1) Abcam product code ab 21083

2) Abcam order reference number 1090459

3) Description of the problem Antibody does not work any more

4) Sample preparation:
Species
Type of sample: cells in culture, U2OS
Sample preparation standard immunofluorescence
Positive control Irradiated cells (10 Gy IR)
Negative control Untreated cells

5) Fixation step
Yes
If yes: Fixative agent and concentration 3.7% Formaldehyde
Fixation time 10 minutes
Fixation temperature Room temperature

6) Antigen retrieval method

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration: 0.5% TritonX 100


8) Blocking agent (eg BSA, serum…):
Concentration 1% BSA
Blocking time 30 minutes
Blocking temperature Room temperature

9) Endogenous peroxidases blocked?
Endogenous biotins blocked?

10) Primary antibody (If more than one was used, describe in “additional notes”) : 53BP1 (abcam) and yH2AX (millipore)
Concentration or dilution 53BP1 1:500, yH2AX 1:250
Diluent buffer 0.5% TritonX 100
Incubation time 2.5 hours room temperature or Overnight at 4 degrees (tried both conditions)

11) Secondary antibody:
Species: Donkey
Reacts against: Rabbit
Concentration or dilution 1:500
Diluent buffer 0.5% TritonX 100
Incubation time 1 hour room temperature
Fluorochrome or enzyme conjugate Alexa Fluor 594

12) Washing after primary and secondary antibodies:
Buffer 0.5% TritonX100
Number of washes 3

13) Detection method Immunofluorescent microscope (Leica)

14) How many times have you run this staining? 4
Do you obtain the same results every time? No, obtained same results in June and now it stopped working
What steps have you altered to try and optimize the use of this antibody? Increasing concentration of Ab, increasing incubation times, permeabilizing with alternative reagents, increasing exposure time on microscope, increasing dosage of IR


Thanks for your help!

Best regards,

Read More
Answer

Thank you for taking time to complete our questionnaire. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. I cannot explain why the activity of the antibody diminished over the time you had it.

One thing I would check is that you do not have a self-defrosting freezer. These automatically defrost, raising and lowering the temperature, forcing the antibody through freeze-thaw cycles, potentially decreasing its activity.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial. There is only one thing I would point out which may be worth taking into consideration. You have used 0.5% TritonX in the antibody diluent as well as the wash buffers. This as well as the permiabilisation step may be too strong. I would use one 10 minute permiabilisation step (using 0.2% Triton X in PBS) at room temperature. Following this I would only use 0.1% Tween 20 in your diluent buffers. You did not mention explicitly but are you using PBS or TBS for the diluent buffers?

If you would like, I can send you a new vial of ab21083 to be sent to you to see if this makes a difference in your staining? If you would like for me to arrange this please could you confirm I have the correct addressbr/>Alternatively, if you would prefer I can offer a credit note, or refund in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More

Question
Answer

Thank you for contacting us yesterday and sorry for the delay in getting back to you.

I am sorry to hear that you have been having some difficulty in using the Anti-53BP1 antibody (ab21083). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As we discussed on the phone, I am attaching our questionnaire so that I can gather further information regarding the samples tested and the protocol used. Once I have receive the completed questionnaire, I will look at the protocol and see if there are any suggestions I can make that may improve the results.

If you could include some images of the staining when the antibody worked and how the latest experiments have been that would be very helpful.

This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

Read More

Question
Answer

Thank you for your inquiry.

Unfortunately, we are not able to release the immunogen sequence for this particular antibody as it is proprietary information. However, I can tell you that it falls within the carboxy-terminus of h53BP1.

We aim to provide as much information as possible to our customers, so I am sorry that this has not been possible on this occasion.

Should you have any further questions, please do not hesitate to contact us.

Read More

Question

Dear Technical, please find attached antibody data for ab21083 , and advise what your suggestion maybe.

Thanks

Cat.#: ab21083
Lot. # GR56537-11
1. Order details:
- Antibody storage conditions (temperature/reconstitution etc)
-20oC
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
No band
3. On what material are you testing the antibody in WB?
- Species: human cancer cells
- Cell extract or Nuclear extract: Nuclear extract
- Purified protein or Recombinant protein: Purified protein
4. The lysate
- How much protein was loaded: 20ul
- What lysis buffer was used: NP-40 lysis buffer
- What protease inhibitors were used: cocktail PMSF
- What loading buffer was used: SDS-PAGE loading buffer 5X
- Did you heat the samples: temperature and time: yes ,95o, 8min
5. Electrophoresis/Gel conditions/ Transfer conditions
- Reducing or non reducing gel: non reducing
- Gel percentage : 10% or 15%
- Transfer conditions: wet transfer 300mA 90min

6. Blocking conditions
- Buffer: TBST
- Blocking agent: milk, BSA, serum, what percentage: no-fat milk 5%
- Incubation time:90min
- Incubation temperature RT:
7. Primary Antibody
- Specification (in which species was it raised against): Hu Ms
- At what dilution(s) have you tested this antibody:
- What dilution buffer was used: TBST
- Incubation time: over night
- Incubation temperature: 4oC
- What washing steps were done: TBST 10minx3
8. Secondary Antibody
- Specification (in which species was it raised against)? rabbit
- At what dilution(s) have you tested this antibody:
- Incubation time:2h
- Wash steps: TBST 10minx3
- Do you know whether the problems you are experiencing come from the secondary?
9. Detection method
ECl, ECl+, other detection method: ECl+ santa
10. Background bands
- Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):
yes, we have run a “No primary” control
- Is the blocking step sufficient? Yes , 2h RT
- Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps)
- At what size are the bands migrating? Could they be degradation products of your target?
- Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)
11. Did you apply positive and negative controls along with the samples? Please specify.
12. Optimization attempts
- How many times have you tried the Western? 3
- Do you obtain the same results every time e.g. are background bands always in the same place?
No bands
- What steps have you altered?

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hearthe customer has haddifficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

As far as I know, Biomoles have been made aware that they are not an authorized distributor of Abcam products. Regrettably, I am sorry this means we are not able to provide replacements or credit notes for faulty items purchased and resold by Biomoles. However, we are pleased to help as much as we can with technical support. Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details:

1. I recommend to check the amount of protein loaded. Try a protein assay on the sample and load 20 -30 ug of total protein per lane of the gel to ensure there is enough protein for detection.

2. Please confirm what type of cancer cells are being tested? What positive controls have been used? For example, HeLa whole cell lysate would be a suitable positive control.

3. Could you confirm if the transfer of protein to the membrane and quality of the sample has been assessed using a loading control?

4. Please confirm what dilutions of antibody have been tried? This may require some optimization.

5. Is the secondary antibody working well with other antibodies?

I hope this information is helpful. Thank you for your cooperation and for your understanding of our policy regarding unofficial distributors. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (MRC5VA)
Loading amount
15 µg
Specification
MRC5VA
Gel Running Conditions
Reduced Denaturing (8%)
Blocking step
Milk as blocking agent for 14 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Apr 19 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (T lymphocyte)
Specification
T lymphocyte
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% triton
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Nov 29 2010

Question
Answer

Thank you for your enquiry. We do notice that there is some size discrepancy between the calculated MW vs. observed MW with the ab21083 antibody. This product has been tested in house on Hela cell lysate and the antigen that was used to generate the antibody by ELISA and WB. We also found that the peptide blocks antibody detection by WB. Based on our testing and data collected we concluded and feel confident that the antibody is detecting the 53BP1 protein. I have included PubMed IDs for several publications which describe detection of the 53BP1 protein by WB. The literatures indicate that 53BP1 protein migrates significantly slower than its predicted size. PubMed ID: 11238909 11134068 9748285 15840649 This product is one of our top selling antibodies and we have not received complaints regarding detection of the incorrect protein. This is currently all the information we have available. But if we do happen to obtain more data, we will be sure to forward you the information.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa and SK-N-SH)
Specification
HeLa and SK-N-SH
Fixative
Paraformaldehyde

Dr. Kirk Mcmanus

Verified customer

Submitted Jul 12 2006

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

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