Overview

  • Product name

  • Description

    Mouse monoclonal to Geminin
  • Host species

    Mouse
  • Tested applications

    Suitable for: ELISA, IHC-P, ICC/IF, WB, Flow Cytmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment, corresponding to amino acids 110-209 of Human Geminin (AAH05185) with a proprietary tag.

  • Positive control

    • Partial tagged recombinant Geminin; Geminin transfected 293T cell lysate; Human lymph node; Human placenta; HeLa cells.
  • General notes

    This product was changed from ascites to tissue culture supernatant on 22 May 2019. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab104306 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
ELISA Use at an assay dependent concentration. Detection limit for recombinant tagged Geminin is approximately 0.03ng/ml when used as a capture antibody.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 24 kDa.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function

    Inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). It is degraded during the mitotic phase of the cell cycle. Its destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle.
  • Sequence similarities

    Belongs to the geminin family.
  • Developmental stage

    Absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition.
  • Information by UniProt
  • Database links

  • Alternative names

    • DNA replication inhibitor antibody
    • Gem antibody
    • GEMI_HUMAN antibody
    • Geminin antibody
    • Geminin DNA replication inhibitor antibody
    • GMNN antibody
    • OTTHUMP00000039393 antibody
    • RP3 369A17.3 antibody
    see all

Images

  • Anti-Geminin antibody (ab104306) at 5 µg/ml + Immunogen at 0.2 µg

    Predicted band size: 24 kDa



    The tagged immunogen has a predicted MWt of 37 kDa.

    This image was generated using the ascites version of the product.

  • All lanes : Anti-Geminin antibody (ab104306) at 5 µg/ml

    Lane 1 : Geminin transfected 293T cell lysate
    Lane 2 : Non-transfected 293T cell lysate

    Lysates/proteins at 25 µg per lane.

    Predicted band size: 24 kDa



    This image was generated using the ascites version of the product.

  • ab104306, at 3 µg/ml, staining Geminin in formalin-fixed paraffin-embedded Human lymph node by Immunohistochemistry.

    This image was generated using the ascites version of the product.

  • ab104306, at 1µg/ml, staining Geminin in formalin-fixed paraffin-embedded Human placenta by Immunohistochemistry.

    This image was generated using the ascites version of the product.

  • ab104306, at 10 µg/ml, staining Geminin in HeLa cells by Immunofluorescence.

    This image was generated using the ascites version of the product.

  • Overlay histogram showing HeLa cells stained with ab104306 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab104306, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    This image was generated using the ascites version of the product.

  • Immunofluorescence analysis of Caki-2 (Clear cell renal carcinoma) cells, staining Geminin with ab104306.

    Cells were fixed with formaldehyde, permeabilized with 0.25% Triton X-100 and blocked with 1% BSA for 30 minutes at 25oC. Primary antibody was concentrated using ab102778 Antibody Concentration Kit and conjugated to Rhodamine using ab102915 EasyLink Rhodamine Conjugation Kit. Samples were incubated with conjugated primary antibody (20 μg/ml in diluent) for 2 hours at 25oC.

    This image was generated using the ascites version of the product.

    See Abreview

References

This product has been referenced in:

  • Kondrashova O  et al. Methylation of all BRCA1 copies predicts response to the PARP inhibitor rucaparib in ovarian carcinoma. Nat Commun 9:3970 (2018). Read more (PubMed: 30266954) »
  • Kondrashova O  et al. Secondary Somatic Mutations RestoringRAD51CandRAD51DAssociated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma. Cancer Discov 7:984-998 (2017). Read more (PubMed: 28588062) »
See all 3 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question
Answer

Thank you for contacting us.

The following protocol was used with ab104306 for ICC/IF:

Preparation of slides:

1. Grow cultured cells on sterile glass cover slips at 37°C overnight.

2. Wash sample with PBS twice.

3. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS).

Step-by-step procedure:

1. Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.

2. Wash cells 3 times with PBS.

3. Incubate cells for 1 hour with normal goat blocking serum (1:20 in PBS).

4. Introduce primary antibodies (in appropriate dilutions) to the sample.

5. Incubate for 4 hours at room temperature or at 4°C overnight.

6. Wash with PBS for 5 minutes. Repeat 5 times.

7. Incubate cover slips in fluorescein-conjugated secondary antibodies in 5% normal blocking serum in PBS in a dark humidity chamber at 4°C for 1 hour.

(Perform all subsequent washes under dim and ambient light source.)

8. Wash sample thoroughly with PBS. Each wash lasting 5 minutes. Repeat 6 times.

9. Counter stain sample with DAPI at room temperature for 30 minutes.

10. Wash sample with PBS. Each wash lasting for 2 minutes. Repeat 3 times.

11. Mount sample by inverting them onto mounting medium on glass slides.

12. Store slides in dark at 4°C.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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

Dr. Melody Jimenez

Verified customer

Submitted Feb 05 2013

Answer

Thank you for your reply.

As I mentioned in my previous email, we do not have sample sizes of our products. Instead we guarantee them, under our Abpromise to work as stated on the datasheet. If for whatever reason they fail to work, then we will replace or refund the antibody for you.

For ab104306 we guarantee to work on human tissue in the following applications: ELISA, IHC-P, ICC/IF, WB. If you wish to try the antibody on a different species, then you can take advantage of our 100% Abreview testing Discount program:

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount



If you would like to take advantage of the testing discount program or if there is anything else I can help you with, please let me know.

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Answer

Thank you for contacting Abccam. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

If the product is to be used in an untested species or application, you may be eligible for our testing discount program if the antibody has not yet been purchased. Please contact our Scientific Support team by replying to this email prior to purchase for more information.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Read More

Answer

I'm glad to hear it. If you do have any further questions please do not hesitate to contact me again.

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

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Question
Answer

I am sorry that the protocol details were not explained clearly enough. I will try to explain as best I can, if it does not make sense please let me know and I'll try to rephrase.

You would take your antibody (concentration of 0.36 mg/mL), you are hoping to achieve a concentration of at least 1 mg/mL to use it with theconjugation kit. In order to do this, you could concentrate all the antibody you will be provided with:

100 ug of antibody at 0.36 mg/mL, gives you 277.7 uL in total. This therefore needs to be concentrated by about a factor of 3 to get the desired concentration. You would therefore load the antibody into the spin cartridge. Spin (start with no more than 1 minute at no more than 15,000g), this will give you an idea of how quickly the solution will be removed. Continue to spin (checking after every minute or less or centrifugation) until you have removed ˜185 uL of solution. You should therefor be left with ˜92 uL of antibody at ˜ 1mg/mL.

If you would like to concentrate only a portion of the antibody, the same process can be followed but reducing the volume added and the volume discarded. Be very careful however to not let the antibody come to complete dryness (try not to reduce the antibody volume to below50-100 uL).

I hope this has helped to clarify the procedure. If you have any further questions, please do not hesitate to contact me again.

Read More

Question

Dear xxx,

Thank you very much for your response.
I'll be happy to receive the ab96895 secondary Antibody as an alternative to the one currently used to the adress indicated below:

When do you think this could arrive to the school?

I appreciate the information provided regarding with the new set of Ab's that I'm planning to acquire and their compatibility with the conjugation link. I'll be looking forward to your response once the team go back to you.

In answer to your questions:

S phase: I've successfully identified the S phase cells by intercalating an analogue of thymidine within the nascent DNA, for doing so, I've used a kit for cell proliferation from the Life Technologies Company and I detect the newly synthesized DNA by means of fluorescent microscopy. This process is very straightforward, I just need a highly proliferative cell culture and to prepare a solution for it to react with and that's pretty much it!. At the beginning of the experimental design of my PhD I decided to label only G1 and S phase cells and therefore to determine G2 and M phase cells by exclusion; however, due to the lack of specificity in my ICC system I will definitely need to label G2 cells to be sure to be sure which is the developmental state of each single cell within my slide.

Please notice that I haven't run the simultaneous labelling of cells (G1 and S) within the same slide so there is no chance for the S phase labelling reagents to alter the results I've got for the G1 labelling procedure. I decided first to optimize each ICC individually and then combine them all together.

Regarding with the progression of the experiments, I have stained 2 more cell lines onto the growth surface of the plates (LNcap and A-498, prostate and renal cell lines respectively) with no satisfactory results yet. (Unfortunately I haven't got any HeLa specimen so far as my collegues work with GU cell lines mainly).

Thank you in advance,

Looking forwar to hearing from you,

Read More
Answer

Thank you for confirming that information. I have arranged for thenew secondary antibody (ab96895) to be sent out to you directly (to the address specified in your last email). The new order number is xxxx(FOCRxxxxx). This should reach you later on today or tomorrow.

I also now have some more input from the lab regarding the compatibility of the anti-Greminin antibody (ab104306)with the Rhodamine conjugation kit. The lab have said thatthey recommend using antibody concentrations of 1-4 mg/mL, asthis generally give the optimal conjugation results. With the antibody being at 0.36 mg/ml, ifused withtheRhodamine Kit, we would expect to not obtain satisfactoryresults as the reactive chemicals would be too dilute.

We would therefore recommendthat the antibody be concentrated prior to carrying out the conjugation procedure. This can be done using the antibody concentration kit, https://www.abcam.com/antibody-concentration-kit-3-columns-ab102778.html.It is worth having a look at the FAQs relating to the conjugation kits we offer which can be accessed from the link below:

https://www.abcam.com/index.html?pageconfig=resource&rid=13156

The antibodies ab104306 and ab81032 would both be compatible with the kit but if considering using any other antibodies with the kit it is important to check that no substances that are incompatible with the kit are present in the solution (Question 5).

I hope this information has been of help. I look forward to hearing how you get on with the newsecondaryantibody. I'll keep my fingers crossed that the experiment works!

Read More

Answer

I'm sorry for the delay in getting back to you, we have been closed over the Easter holidays.

I have had a look at the secondary antibodies you have suggested as trying as an alternative to the one currently used (ab60314). All seem fine for the application you hope to use. If you would like I can send this out to you free of charge. Unless you have a preference for a different one, I'd send out ab96895.You mentioned that you were in Ireland? Would you still like for this replacement secondary antibody to be sent to the address I have for you:

xxxx


I am not sure how the antibody concentration of anti-Geminin antibody ab104306 would affect the use in conjunction with the conjugation kit (ab102915). If will forward this query to the source of this kit and will let you know as soon as I have a response. As you will be conjugating directly, I would suggest that using an isotype control would be quite important asyou are not able to do a "no primary" control experiment to ascertain the non-specificity observed.Mouse monoclonalab81032 should be suitableand would becompatiblewiththe conjugationkit.

Can I ask, how are you going to detect the S phase (with Alexa Fluor 647), are you using another antibody? In which case, what antibodies do you intend to use?

How have your subsequent experimentsprogressed? Have you used the HeLa cellswith adherent slides?

I hope this information has been of help.I look forward toreceivingyour response.

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