Overview

  • Product name

  • Description

    Rabbit polyclonal to CDT1/DUP
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human CDT1/DUP aa 500 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab93742)

  • Positive control

    • This antibody gave a positive signal in both human brain and liver tissue lysates.
  • General notes

     This product was previously labelled as CDT1

     

Properties

Applications

Our Abpromise guarantee covers the use of ab83174 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 Use a concentration of 1 µg/ml. Detects a band of approximately 73 kDa (predicted molecular weight: 60 kDa).
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function

    Cooperates with CDC6 to promote the loading of the mini-chromosome maintenance complex onto chromatin to form the pre-replication complex necessary to initiate DNA replication. Binds DNA in a sequence-, strand-, and conformation-independent manner. Potential oncogene.
  • Sequence similarities

    Belongs to the Cdt1 family.
  • Developmental stage

    Present during G1 and early S phase of the cell cycle. Degraded during the late S, G2, and M phases.
  • Domain

    The PIP-box K+4 motif mediates both the interaction with PCNA and the recuitment of the DCX(DTL) complex: while the PIP-box interacts with PCNA, the presence of the K+4 submotif, recruits the DCX(DTL) complex, leading to its ubiquitination.
  • Post-translational
    modifications

    Ubiquitinated by the DCX(DTL) complex, also named CRL4(CDT2) complex, in response to DNA damage, leading to its degradation. Ubiquitination by the DCX(DTL) complex is necessary to ensure proper cell cycle regulation and is PCNA-dependent: interacts with PCNA via its PIP-box, while the presence of the containing the 'K+4' motif in the PIP box, recruit the DCX(DTL) complex, leading to its degradation. The interaction with GMNN protects it against ubiquitination.
    Phosphorylated by cyclin A-dependent kinases which results in the binding of CDT1 to the F-box protein SKP2 and subsequent degradation. Binding to GMNN is not affected by phosphorylation.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Database links

  • Alternative names

    • CDT 1 antibody
    • cdt1 antibody
    • CDT1_HUMAN antibody
    • Chromatin licensing and DNA replication factor 1 antibody
    • DNA replication factor antibody
    • DNA replication factor Cdt1 antibody
    • Double parked antibody
    • Double parked Drosophila homolog of antibody
    • Double parked homolog antibody
    • DUP antibody
    • Retroviral integration site 1 antibody
    • Retroviral integration site 2 antibody
    • Retroviral integration site1 antibody
    • Retroviral integration site2 antibody
    • RIS 2 antibody
    • RIS2 antibody
    see all

Images

  • All lanes : Anti-CDT1/DUP antibody (ab83174) at 1 µg/ml

    Lane 1 : Human liver tissue lysate - total protein (ab29889)
    Lane 2 : Human brain tissue lysate - total protein (ab29466)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 60 kDa
    Observed band size: 73 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 1 minute


    The 73 kDa band observed is also comparable to the molecular weight seen with other commercially available antibodies to CDT1/DUP.

  • ICC/IF image of ab83174 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab83174, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.

References

This product has been referenced in:

  • Jimenez-Hernandez M  et al. Exploring the spectroscopic differences of Caki-2 cells progressing through the cell cycle while proliferating in vitro. Analyst N/A:N/A (2013). Read more (PubMed: 23640135) »
  • Wong VC  et al. S-phase sensing of DNA-protein crosslinks triggers TopBP1-independent ATR activation and p53-mediated cell death by formaldehyde. Cell Cycle 11:2526-37 (2012). Read more (PubMed: 22722496) »
See all 3 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Caki-2 (Clear cell renal carcinoma))
Permeabilization
Yes - 0.25% X-Triton in PBS
Specification
Caki-2 (Clear cell renal carcinoma)
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Formaldehyde

Dr. Melody Jimenez

Verified customer

Submitted Feb 11 2013

Question


Thank you for the reply.
In answer to your questions:
1. Unfortunately I have no data supporting the expression levels of
the CDT1 in this particular cell line so it's definitely worthwhile to
stain HeLa cells and see whether the weak fluorescent signal arises
due to the low concentration of CDT1 within my cells.
2. The cell viability is checked by means of the trypan blue exclusion
method, about 1-2% of cells are dead as they don't efflux effectively
the dye, therefore I've confirmed in every sample prep that the cells
are viable. Also, the cells are seeded at a concentration of 40,000
cells/ml (concentration that has shown to give place to a log growth).
The ration cells per area is: 12,500 cells/cm2 (which has demonstrated
by previous studies that enable the log growth of the cells).
3. Unfortunately the secondary antibody hasn't been used with another
primary antibody and the primary antibody hasn't been detected by
another secondary antibody -this is the only system I currently have,
but I do see the point of checking the actual effectiveness of both so
I'll try to get some aliquotes from my collegues and see what sort of
staining profile I get-.
4. I'll run the experiment you suggested next week (as soon as I get
some HeLa cells). And I'll also grow the cell line of interest
(Caki-2) and perform the staining on the glass they've growth on so we
can conclusively know whether the trypsin is having some detrimental
effect on the staining.
At the moment I'm staining my last batch of samples (fixed with 50%
methanol and 50% acetone). The samples are going to be incubated with
the primary antibody overnight at 4C and hopefully the specific signal
from the positive control will be better.
Best regards,
PS. As I'm not an expert on the art of performing ICC nor in biology,
I've been reading about those things that might potentialy affect the
staining and I've noticed that the majority of the antibodies are
aliquoted and stored either at -20 or -80 C. When I first got my
antibodies, I followed the storage instructions for all of them;
therefore I aliquoted both: the primary antibody (ab83174) and the
Rabbit Isotype Ctrl (ab37406) and then I stored them at -20 C. I also
followed the storage instructions for the secondary antibody (ab60314)
shown in both the datasheet that the antibody came with and the Abcam
website for this particular antibody so I've kept it at 4C. I wonder
if perhaps there is a mistake on the actual storage instructions and
therefore this antibody has gone bad as it's been kept @ 4C for about
4 months by now?
Sorry for the silly question but at this point I'm trying to track any
potential failure in the whole system as I have not got a successful
staining profile yet.
Thank you in advace.

Read More
Answer

I think its good that you are checking the cell viability and using HeLa may provide more information as to if it is the cell strain or the antibodies that may be contributing to the problems. I agree that checking both the primary and secondary antibody is excessive but if you have access to an alternative secondary antibody it may be worthwhile trying. The activity of the secondary antibody should not have been compromised as you are storing it as we have recommended (and I have checked that these guidelines are correct). The antibody should also be stored in the dark, as should ab37406. Ab37406 and ab83174 should be aliquotted (we usually recommend no lower than 10 uL) and stored at -20 or -80. After thawing an aliquot this should not be refrozen but can be storedat 4degreesfor a few weeks if some is left over after your experiment.


Let me know how you get on with your experiments. I'll be crossing my fingers for you.

Read More

Question

Hi xxxx,

Firstly I'd like to thank you for all the suggestions you've made so far, it's been a very nice Abcam experience and the better technical support I've ever got. So, thank you again.

You are right, now we now that there is definitely occuring non-specific binding of both antibodies.

Cytospinning or growing the cells onto the substrate.
The renal cell line I'm working with is called Caki-2 and it is adherent. Even though the cells DO grow onto the substrate (CaF2 slides without any sort of coating) they tend to grow very close to each other -even after just 24 hours of being seeded at a low cell density - making clumps of cells that are not useful for me any more as I'm planning to collect spectroscopic data from isolated "single-cells" before actually performing any ICC on them (which takes about 2 days) >> that's also the main reason for which I fix the cells and leave them dry (water is a strong absorber of IR radiation).

The special substrate that I need for collecting the spectra isn't particularly "friendly" with the cells, therefore some toxicity might arise from it. Putting a coating of either L-lysine or some other thing onto the surface would mask further slight spectral variations due (for example) cytotoxicity of anticancer drugs or cell-cycle (that I'm planning to detect). As the nature of the substrate isn't completely suitable for growing the cells onto it I need therefore to cytospin them down onto the surface of something IR inactive (in this case CaF2 windows) that enable me to collect useful spectral information without actually interfering with the inherent chemistry of my treated cells.


Fixative
The reason for which I chose to use PFA as cell fixative instead methanol fixation relies on the fact that by using the later one some lipids from the membrane may be removed (and by means of spectroscopy it will be not ideal to have a cell without a complete membrane).
As I'm planning to collect IR data from my samples and then ICC stain them I can not keep them in a hydrated form and perform ICC immediately.

However, what I'll do following your suggestion will be to run a parallel ICC test on the next batch of samples with the same concentration of antibodies and see whether the fixation process is a contributing factor for giving place to the non-specific binding. If the results are definitely better with methanol then I will modify the overall protocol in order to get the most out of the ICC staining (which is the important bit at the moment!!). =)

I will also fix the cells after they have been cytospun as you suggested.
You were right, the antibodies have always been diluted in 1% BSAin PBST.

I'll go back to you once I get the next experimental results -hopefully they will be brilliant!-

Best wishes,

xxxxxx

Read More
Answer

Thank you for providing such a full explanation of what you are trying to achieve. I can now understand exactly why you have chosen the technique that you have.

I think the experiments you are planning to do next will be very useful in determining how best to proceed with the staining and more fully understandingwhere the non-specificity iscoming from.I too hope they will be brilliant! I don't knowif you areplanning on it yet but I would suggest keeping at least oneof your test withoutair dryingyourcells(i.e keeping them hydrated throughout), just to seeif thisimproves the non-specificity seen with the secondary and isotype control antibodies. If it does, it would make youractual experimentdifficult to measure theIR butI think it is most important at the moment toget the specific staining. Once this has been achieved, the optimisationof how toperform the actual experiments can be done.

I wish you all the best with the new experiments and look forward to hearing how you get on.

Read More

Question

Dear xxxx,

I contacted you a few weeks ago as I'm having having some problems for stainng my samples with the following ICC system:
Primary antibody: Anti-CDT1 antibody (ab83174)
Secondary Antibody: Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Chromeo™ 488) (ab60314)
Control Isotype: Rabbit IgG FITC - Isotype Control (ab37406)
I performed the ICC staining of my samples last week by following all the useful suggestions you made in order to optimize the protocol and unfortunately I still get signal coming from the controls.
Please find attached to this email a .ppt file that describes the experiments I conducted and the images collected after staining my samples.
I wonder if you could provide some advice in terms of what to do under these circumstances.
Any information will be much appreciated.

The modifications of the protocol were the following:

Formalin fixation: Yes. Time: 15 minutes (rather than one hour).
Blocking time: 45 minutes with 10% Goat serum in 1%BSA in PBS (rather than 30 minutes).
Incubation time for each antibody: 1 hour at room temperature (rather than overnight at 4C)
Dilution of the primary antibody: 5 ug/ml (as recommended).
Dilution of the secondary antibody: 1:1500 and 1:2000 (1.4 and 1 ug/mL respectively rather than 4ug/mL).

Also, my supervisor suggested to hydrate the samples before doing the actual ICC staining by incubating them with PBS for 30 minutes, I wonder if perhaps this was not a good idea and may be a contributing factor for giving place to the misbinding?

Best wishes,

Read More
Answer

Thank you for getting back to me with your new results. It is good that we can now say conclusively that there is non-specific binding of both the secondary antibody and of the isotype control antibody.

Following the protocol changes, I do not exactly know why this non-specific nuclear staining is persisting but I have a few suggestions to add that may be beneficial.

I am not an expert in using cytospin by any means so I have been having a read about it. It seems that it is generally recommended to first spin your suspension, then fix the cells on the slides. The following reference may be of help to you:

http://www.ihcworld.com/_protocols/histology/cytospin.htm

http://sciencepark.mdanderson.org/labs/tang/cytospin4web.html

It may also be beneficial to fix immediately, then stain immediately. I would not allow your cellsto dry out at any stage.The only Abreview we have of ab83174 having been used in ICC used PFA fixation and some non-specific staining of the cytoplasm was also seen (which seems to be what you are seeing). We have charaterised this target using methanol fixation (refer toab83174 datasheet). It maybe worthwhile to try this in order to improve the staining.

Is there a reason why you are using cytospin? If your cells are adherent you could just grow the cells on glass slides directly as is described in the following protocol:

https://www.abcam.com/index.html?pageconfig=resource&rid=11417

I have a further question. What diluent are you using for your antibodies? I would suggest still usingthe same asyouwere (1% BSAin PBST).Are you performingany washing steps? Following each incubation I would wash theslide with PBS 3 times for 5 minutes.

If you have any questions in regards to my suggestions please do let me know.

Read More

Answer

Sorry for the delay in getting back to you and thank you for all that information. It makes it much easier to understand what is going on and what has been tried previously.

I think the next step you are suggestingare good. The blocking buffer and antibody diluent you are using are both what were used in performing inhouse testing with this antibody. Additionally, performing the following controls:

1. isotype control only

2. secondary antibody only (no-primary control)

3. primary antibody+secondary antibody

you will see more accurately what element is contributing to the non-specific signal.

There are a few things I would add though.

1. for fixation with formalin we would normaly say 10-15 minutes would be enough. The danger in prolonging the fixation is that the epitope that the antibody recognises can be masked, requiring antigen retrieval to be employed. It can also lead to increased non-specific binding. I would therefore suggest reducing the fixation time.

2. the recommended dilution for the secondary antibody you are using is 1/1000 to 1/2000. This is equivalent to 1-2 µg/mL. I would therefore reduce the concentration used from 4 µg/mL to 1 µg/mL, which will hopefully help in reducing the non-specific signals observed.

Additionally, may I ask how thedilutionof the isotype control was performed?

The isotype that you are using I would not specifically recommend as a control for the experiment you are performing. As the antibody is already FITC conjugated, if it does bind non-specifically and the secondary also binds, you will get an amplification of the signal over and above what the actual primary antibody non-specificity would yield. I would either suggest using a primary antibody that is directly FITC conjugated, or an isotype control such as ab27478.

Ihope the suggestions I have made improve the results seen so far. I look forward to hearing how you get on.

Read More

Answer

Thank you for contacting us and reporting the problems you have encountered in using the Anti-CDT1 antibody (ab83174) and rabbit IgG isotype control (ab37406).


As discussed over the phone, in order to provide the most suitable suggestions I would like to understand the procedures you are using more fully. I have therefore attached a questionnaire to collect more information. If you could also include some images of the results you have been obtaining that would be very useful.


Could you also please elaborate on the following points:


1. Have you performed a "no-primary" control and if so what was the result?


2. Have you tried incubating the isotype control only with the sample? If so what was the results?


With this information hopefully I will understand more fully what may be contributing to the unexpected results.


I look forward to receiving your reply.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
Yes - 0.5% Triton X100
Specification
HeLa
Fixative
Paraformaldehyde

Dr. Kirk Mcmanus

Verified customer

Submitted Mar 15 2011

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