Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)

Reviews (2)Q&A (4)References (9)

Overview

  • Product name
    Anti-Cyclin B2/CCNB2 antibody [X29.2]
    See all Cyclin B2/CCNB2 primary antibodies
  • Description
    Mouse monoclonal [X29.2] to Cyclin B2/CCNB2
  • Host species
    Mouse
  • Specificity
    Reacts with most cyclin Bs.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Xenopus laevis
  • Immunogen

    Full length protein corresponding to Xenopus laevis Cyclin B2/CCNB2.

  • General notes

     This product was previously labelled as Cyclin B2

     

Properties

Applications

Our Abpromise guarantee covers the use of ab18250 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
IHC-P Use a concentration of 1 µg/ml. 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 a concentration of 2 µg/ml. Predicted molecular weight: 45 kDa. The antibody may give a high background if the concentration of the antibody is above 2µg/ml.
Flow Cyt Use 1µg for 106 cells.

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

 

Target

Images

  • Western blot - Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)
    Western blot - Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)This image is courtesy of an anonymous Abreview
    All lanes : Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250) at 2 µg/ml

    Lane 1 : Whole cell lysate of Mitotic Hela cells (arrested by the spindle checkpoint due to depolymerization of microtubles by exposure to Nocodazole)
    Lane 2 : Whole cell lysate of Interphase Hela Cells, forced to exit mitosis by exposure to a Cdk1 inhibitor (exit from mitosis results in the degradation of Cyclin B2/CCNB2)

    Secondary
    All lanes : HRP-Conjugated Goat anti-Mouse diluted 1/20000

    Developed using the ECL technique.

    Predicted band size: 45 kDa
    Observed band size: 45 kDa
    Additional bands at: 90 kDa (possible non-specific binding)


    Exposure time: 25 minutes


    The gel was denaturing. Blocked with a 5% milk solution for 2 hours at 24°C.

    The primary antibody was diluted in 5%Milk/Tris-Buffered Saline/0.05%Tween20 and incubated for 16 hours at 4°C.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)
    IHC image of ab18250 staining in human cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18250, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Flow Cytometry - Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)
    Flow Cytometry - Anti-Cyclin B2/CCNB2 antibody [X29.2] (ab18250)
    Overlay histogram showing HeLA cells stained with ab18250 (red line). The cells were fixed with 100% 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 (ab18250, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

References

This product has been referenced in:
  • Möckel MM  et al. Xenopus laevis Kif18A is a highly processive kinesin required for meiotic spindle integrity. Biol Open 6:463-470 (2017). Read more (PubMed: 28228376) »
  • Dupré AI  et al. The greatwall kinase is dominant over PKA in controlling the antagonistic function of ARPP19 in Xenopus oocytes. Cell Cycle 16:1440-1452 (2017). Read more (PubMed: 28722544) »
See all 9 Publications for this product

Publishing research using ab18250? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Question

Thanks for review this data. We have rerun the test again according to your below recommendations, we were still unable to achieve a good outcome. I am attaching the data with email for your review again. we hope to hear of your advise, and whether a different lot might be accessible.

We apologize for any inconveniences.

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Answer

Thank you for your reply. I am sorry that the antibodies are still not producing good results in WB. We do not currently have new lots available for either product, so I have issued you a refund (Credit Note: ***). To redeem this refund, please have your purchasing agent contact our accounting department at us.credits@abcam.com. Please let me know if you have any further questions.

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Question

Attached please find the antibody ab18250 & ab75743wb questionnaire answer sheets. We did not observe an optimal results over these two antibodies. we would look forward to any suggestion or advice you may have.

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Answer

Thank you for contacting us. I am sorry that these antibodies are giving weak signal in your samples.

For Cyclin B2, I would expect ab18250 to give good staining using the protocol you sent, however this protein is not very highly expressed in normal or cancerous liver. You may find better results testing a positive control such as bone marrow or HeLa cells.

The antibody ab75742 is phospho-specific, so I would not recommend using milk as a blocking agent. Milk contains endogenous phosphatases which can cleave the phosphorylations of interest and results in significantly reduced signal. Switching to BSA may help to improve the strength of your band. Have you done any treatment as a positive control to ensure that this particular phosphorylation is expressed?

I hope this helps, if not, please let me know and I will be happy to help you further.

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Western blot abreview for Anti-Cyclin B2/CCNB2 antibody [X29.2]

 Average
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
abreview image
Application
Western blot
Sample
Xenopus laevis Cell lysate - whole cell (early embryos before MBT)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
1 cells
Specification
early embryos before MBT
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Dr. Jolanta Kisielewska

Verified customer

Submitted Jul 23 2009

Western blot abreview for Anti-Cyclin B2/CCNB2 antibody [X29.2]

 Good
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Hela)
Gel Running Conditions
Reduced Denaturing
Loading amount
10000 cells
Treatment
Nocodazole 4 hours, Mitotic Shake-Off
Specification
Hela
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Read More

Abcam user community

Verified customer

Submitted Nov 27 2007

Question

I' m sending the informations that can be useful to receive suggestions about 18250 Ab. Blocking solution prepared: 3% BSA in PBS 1X Antibody dilution utilized: 1/1000 in blocking solution Waiting your reply, all my best regards.

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Answer

Thank you for your e-mail. I would recommend changing: - the blocking buffer to TBST (tris HCl (pH7.6) with Tween20 0.1%) and trying 5%BSA (1hr) or 5% milk (1hr) -the antibody dilution buffer to TBST only and try also 0.2% milk in the TBST. I think one of those options will work for you. Here is my recipe for the TBST buffer: • TBS 10x (concentrated TBS) -24.23g Trizma HCl -80.06g NaCl Mix in 800ml ultra pure water. pH to 7.6 with pure HCl. Top up to 1L. • TBST For 1L: 100ml of TBS 10x + 900ml ultra pure water + 1ml Tween 20 Please let me know if you require further assistance,

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Question

Abcam Order Number: 73632 Ab18250 Batch number: 144180 This is to inform you on the reactivity of ab18250 raised against Xenopus Cyclin B2. Initial attempts to recognize cyclin B2 from total cell extracts deriving from Rana temporaria tissues by immunoblot failed. Therefore, we tested the antibody against a positive control represented by metaphase II blocked Xenopus eggs. According to Gautier and Maller (EMBO J. 1991 Jan;10(1):177-82) these extracts should contain plently of cyclin B2. However, as you can see from the attached image, we were unable to detect any cross-reacting band on WB at the expected molecular wiight, i.e. about 45 kDa according to the Ab18250 data sheet. On the contrary, we got multiple and not-specific bands. Could you please help us to better settle down our immunoblot? Do you advice a different positive control? If no solutions are available should we replace this antibody with a different one's? We are waiting for an answer at your convenience. Sincerely yours,

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Answer

The originating scientist has got back to me about your query. He says that he has found that the antibody may give a high background in an immunoblot if the concentration of the antibody is above 2µg/ml. He says that you should optimise using a range of dilutions and may need to change blocking buffer. I am still waiting for the positive control information,my apologies for the delay,

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