Overview

  • Product name
    Anti-Rad17 (phospho S645) antibody
    See all Rad17 primary antibodies
  • Description
    Rabbit polyclonal to Rad17 (phospho S645)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Chimpanzee, Rhesus monkey, Gorilla, African green monkey, Orangutan
  • Immunogen

    Synthetic phospho peptide (Human) - which represents a portion of human Rad 17around serine 645.

  • Positive control
    • HeLa whole cell lysate treated with 10 Gy IR.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.1% Sodium azide
    Constituents: 0.021% PBS, 1.764% Sodium citrate, 1.815% Tris
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Antibodies that were not phospho-specific were removedby solid phase absorption. Antibodies specific for Rad 17pSer 645 were affinity purified using the phosphopeptideimmobilized on solid support.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab3620 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
WB 1/500 - 1/5000. Detects a band of approximately 85-88 kDa (predicted molecular weight: 77 kDa).
ICC/IF Use at an assay dependent dilution. PubMed: 20368419

Target

  • Function
    Essential for sustained cell growth, maintenance of chromosomal stability, and ATR-dependent checkpoint activation upon DNA damage. Has a weak ATPase activity required for binding to chromatin. Participates in the recruitment of the RAD1-RAD9-HUS1 complex onto chromatin, and in CHEK1 activation. May also serve as a sensor of DNA replication progression, and may be involved in homologous recombination.
  • Tissue specificity
    Overexpressed in various cancer cell lines and in colon carcinoma (at protein level). Isoform 2 and isoform 3 are the most abundant isoforms in non irradiated cells (at protein level). Ubiquitous at low levels. Highly expressed in testis, where it is expressed within the germinal epithelium of the seminiferous tubuli. Weakly expressed in seminomas (testicular tumors).
  • Sequence similarities
    Belongs to the rad17/RAD24 family.
  • Post-translational
    modifications
    Phosphorylated. Phosphorylation on Ser-646 and Ser-656 is cell cycle-regulated, enhanced by genotoxic stress, and required for activation of checkpoint signaling. Phosphorylation is mediated by ATR upon UV or replication arrest, whereas it may be mediated both by ATR and ATM upon ionizing radiation. Phosphorylation on both sites is required for interaction with RAD1 but dispensable for interaction with RFC3 or RFC4.
  • Cellular localization
    Nucleus. Phosphorylated form redistributes to discrete nuclear foci upon DNA damage.
  • Information by UniProt
  • Database links
  • Alternative names
    • CCYC antibody
    • Cell cycle checkpoint protein antibody
    • Cell cycle checkpoint protein RAD17 antibody
    • FLJ41520 antibody
    • HRAD 17 antibody
    • hRad17 antibody
    • R24L antibody
    • Rad 17 antibody
    • Rad 24 antibody
    • RAD1 (S. pombe) homolog antibody
    • RAD1 homolog antibody
    • RAD17 antibody
    • RAD17 homolog (S. pombe) antibody
    • RAD17 homolog antibody
    • Rad17 like protein antibody
    • RAD17, S. pombe, homolog of antibody
    • RAD17_HUMAN antibody
    • RAD17Sp antibody
    • Rad24 antibody
    • Rad24, mouse, homolog of antibody
    • Rad24, S. cerevisiae, homolog of antibody
    • RF C activator 1 homolog antibody
    • RF C/activator 1 homolog antibody
    • RF-C/activator 1 homolog antibody
    see all

Images

  • Sample: Whole Cell Lysate (50µg/lane) from Hela cells treated with 10Gy IR or untreated.

    Rabbit anti-Rad 17 pSer645 (ab3620) used at 1:1,000 dilution

    Sample: Whole Cell Lysate (50µg/lane) from Hela cells treated with 10Gy IR or untreated.

    Rabbit anti-Rad 17 pSer645 (ab3620) used at 1:1,000 dilution

References

This product has been referenced in:
  • Shin MH  et al. ATM-dependent phosphorylation of the checkpoint clamp regulates repair pathways and maintains genomic stability. Cell Cycle 11:1796-803 (2012). Read more (PubMed: 22453082) »
  • Kumar A  et al. Nuclear phosphoinositide 3-kinase beta controls double-strand break DNA repair. Proc Natl Acad Sci U S A 107:7491-6 (2010). WB, ICC/IF ; Mouse . Read more (PubMed: 20368419) »
See all 3 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1048217 (ab63442).

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Answer

I am sorry to hear that the results does not improve. I can offer you another antibody we have following antibodies against the same target

ab63442; https://www.abcam.com/Rad17-phospho-S645-antibody-ab63442.html

ab115876; https://www.abcam.com/Rad17-phospho-S645-antibody-ab115876.html

Please check ab115876 is a mouse polyclonal antibody, you would need anti mouse secondary if you opt for this.

Let me know which ab you would like to try.

Read More

Answer

Well that's true. BSA reduces the background only, it does not have any effect on ab specificity.

Anyway to proceed further in this case, I would like to ask if you are happy in trying a different lot or a different antibody. Alternatively I can also provide you full refund or credit note, which you can use for future purchases. Let me know the option you would like to go for.

Many thanks for your cooperation in this case, I highly appreciate that.

Read More

Answer

Thank you very much for your email.

We use 5% BSA (Sigma) in TBST for blocking and for diluting secondary and primaries. Milk in not recommended for phospho specific antibodies because milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Please be advised that BSA will only reduce the background i.e. membrane dirtiness, it will not effect the antibody specificity. So if the membrane shows high background then I would certainly recommend trying BSA.

The other important point is, whether the cells naturally express the protein or not. I am sorry the info of cell line wasn't included in your email so I can't be sure about it. I would however recommend using Hela lysates as positive control. Are you sing human cell line?
Actin or GAPDH can be used as loading control. Have you tried any of these?

I hope these suggestions will help to improve your results. If the results do not improve please do not hesitate to contact me.

Read More

Question

It will take approximately 5 minutes to complete the questionnaire. Please fill-in all fields so that we can better assist you.

1) Abcam product code ab3620

2) Abcam order reference number or product batch number GR686-4

3) Description of the problem

No signal

4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…) whole cell lysates

Lysis buffer RIPA buffer

Protease inhibitors: Protease inhibitor cocktail

Phosphatase inhibitors Phosphatase inhibitor cocktail

Reducing agent SDS

Boiling for ≥5 min? yes, 10 min

Protein loaded ug/lane or cells/lane 51 ug

Positive control n/a

Negative control n/a

5) Percentage of gel 4-20% gradient gel

Type of membrane PVDF

Protein transfer verified ponceau reagent

Blocking agent and concentration 5% milk in TBST

Blocking time 1 hour

Blocking temperature room temperature

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

Concentration or dilution 1/500

Diluent buffer 5% milk in TBST

Incubation time overnight

Incubation temperature: 4 degree


7) Secondary antibody:

Species:Donkey

Reacts against: rabbit

Concentration or dilution 1/5000

Diluent buffer 5% milk in TBST

Incubation time 1 hour

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate:HRP

8) Washing after primary and secondary antibodies:

Buffer TBST

Number of washes 3 times, 5 minutes each

9) Detection method Chemiluminascence

10) How many times have you run this staining? twice

Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody? no

Read More
Answer

Thank you for your enquiry regarding ab3620 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.

The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying:

ab3620 is a phospho specific antibody. We used 10Gy IR treated Hela cells to test the specificity of this antibody. I would recommend treating your sample cells there by inducing the phosphorylatuion of amino acid chemically or by radiations.

Milk is not recommended as blocking reagent with phospho specific antibodies. Please use BSA.

Please try both of these suggestions and let us know the results; we will be happy to help further.

Read More

Answer

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.



In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.



As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.



All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.



Please could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.



Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Read More

Answer

To our knowledge, these three antibodies have not been used in parallel, which would be required to determine if there are truely differences in the apparant molecular weight of the protein recognized by each antibody. The differences in the reported apparant molecular weights of the proteins detected by the 3 antibodies is likely owing to differences in molecular weight markers that where used, position of the sample relative to the positon of the molecular weight markers in the gel, and size and composition of the resolving gels. The difference in relative mobility of the protein recognized by the goat anti-Rad17 and the phospho specific antibody is certainly within experimental error.

Read More

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