Overview

  • Product name
    Anti-CDKN2A/p16INK4a antibody [DCS50.1]
    See all CDKN2A/p16INK4a primary antibodies
  • Description
    Mouse monoclonal [DCS50.1] to CDKN2A/p16INK4a
  • Host species
    Mouse
  • Specificity
    This antibody shows no cross reactivity with the closely related inhibitors p15INK4b and p18INK4c.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WB, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein corresponding to Human CDKN2A/p16INK4a aa 1-156.
    Sequence:

    MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQ VMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRA GARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEG PSDIPD


    Database link: P42771

  • Positive control
    • WB: HEK-293 and HeLa cell lysates. Flow cyt: HEK-293 cells. IHC-P: Bladder tumor tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab16123 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
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 10 µg/ml.
WB Use at an assay dependent concentration. Detects a band of approximately 16 kDa (predicted molecular weight: 16 kDa).
IP Use at an assay dependent concentration.

ab16123 co-precipitates cdk4/cdk6.

Flow Cyt Use 1µg for 106 cells.

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

Target

  • Cellular localization
    Cytoplasmic and Nuclear
  • Database links
  • Form
    There are 4 isoforms produced by alternative splicing. Isoform 1 also known as: p16INK4a; Isoform 3 also known as: p12; Isoform 4 also known as: p14ARF; p19ARF; ARF.
  • Alternative names
    • CCM2 antibody
    • CDK4 inhibitor p16 INK4 antibody
    • CDK4I antibody
    • CDKN2 antibody
    • CDKN2A antibody
    • Cell cycle negative regulator beta antibody
    • CMM2 antibody
    • Cyclin dependent kinase 4 inhibitor A antibody
    • Cyclin dependent kinase inhibitor 2A (melanoma p16 inhibits CDK4) antibody
    • Cyclin Dependent Kinase Inhibitor 2A antibody
    • Cyclin dependent kinase inhibitor 2A isoform 4 antibody
    • Cyclin dependent kinase inhibitor 2A isoforms 1/2/3 antibody
    • Cyclin dependent kinase inhibitor p16 antibody
    • INK4 antibody
    • INK4A antibody
    • MLM antibody
    • MTS1 antibody
    • Multiple tumor suppressor 1 antibody
    • p14 antibody
    • p16 antibody
    • P16INK4 antibody
    • p16INK4a antibody
    • p19 antibody
    • p19Arf antibody
    • TP16 antibody
    see all

Images

  • All lanes : Anti-CDKN2A/p16INK4a antibody [DCS50.1] (ab16123) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 16 kDa
    Observed band size: 16 kDa
    Additional bands at: 37 kDa, 50 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 12 minutes
  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded bladded tumor tissue labelling CDKN2A/p16INK4a with ab16123 at 10µg/ml.

  • ICC/IF image of ab16123 stained Hepp cells. The cells were 4% formaldehyde fixed (10 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 (ab16123, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) ab150113) 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.

  • Overlay histogram showing HEK293 cells stained with ab16123 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16123, 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 HEK293 cells fixed with 100% methanol used under the same conditions.

    Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

References

This product has been referenced in:
  • Kannappan R  et al. p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo. EBioMedicine 16:224-237 (2017). Read more (PubMed: 28163043) »
  • Fernández-Torrón R  et al. Cancer risk in DM1 is sex-related and linked to miRNA-200/141 downregulation. Neurology 87:1250-7 (2016). Read more (PubMed: 27558368) »
See all 4 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
ELISA
Sample
Human Recombinant protein (Purified recombinant p16 (overexpressed in HEK293))
Specification
Purified recombinant p16 (overexpressed in HEK293)
Type
Sandwich (Capture)
Blocking step
SuperBlock PBS Pierce #37580 as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 10µg/mL

Abcam user community

Verified customer

Submitted Apr 28 2014

Answer

Thank you for contacting us.

Abcam takes product quality seriously and we recently undertook an in-depth study examining storage conditions and antibody performance. After over 13,000 individual ELISA experiments, we have determined that storing our antibodies at various temperatures of up to 45°C for 1 week does notimpact their activity. I would be happy to share a summary of our findings with you; please let me know if you would like to see this.

As a precautionary measure only, we ship our antibodies in packaging with ice packs to provide extra temperature stability in transit. The ice pack may be thawed or even at room temperature when you receive it; please be assured that this is normal and that your product is safe to use. Once you have received the vial, please follow the long-term storage instructions on the datasheet. With the Anti-CDKN2A/p16INK4a antibody [DCS50.1] (ab16123) we would advise aliquotting the productand storing at -20°C.

If you do encounter any problems in using the antibody, our Abpromise to you is that, should our product not work as stated on the datasheet, we will resolve the issue to your satisfaction. Either with helpful advice to optimize your experiment or a replacement or refund of the product. Please contact our knowledgeable Scientific Support staff should you experience any issues while using our products.

I hope this information helps, but please don't hesitate to contact me for additional information or assistance.

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Answer

Thank you for taking time tocontact us.

For ab16123, have you tried running a positive control with HeLa cell lysate? It may be possible that your cell line is expressing a different isoform. In our testing, we do see the predominant band at 16kDa with HeLa cells, but we also see a faint band at 37 kDa which may be the isoform that is predominant in your samples. Or, you may want to do a cellular fractionation with your samples. It does seem that the different isoforms have different locations (cytoplasm, nucleus).

http://www.ncbi.nlm.nih.gov/pubmed/17658461

For ab54210, the 40 kDa band is what has been detected in the lab. I don't see that we have any specific references for this antibody in WB, but we do have some background references from the lab:

1. Hunter, T. 1993. Cell 75: 839-841.
2. Sherr, C.J. 1993. Cell 73: 1059-1065.
3. El-Deiry, W.S., et al. 1993. Cell 75: 817-825.

I hope this information helps. Please contact us with any other questions.

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Answer

Thank you for contacting us.

Unfortunatley we do not calculate the isoelectric point of our research antibodies.

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

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Question
Answer

I am very pleased to hear you would like to accept our offer and test ab16123 in mouse in Flow cytometry. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview formouse in Flow cytometryand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Question

Good afternoon, we are contacting the Abcam technical support for a feedback of the product Ab16123. We already contacted you on October, 13rd. At first, we wrote to you because, after trying and trying so many times, we realized that the Ab16123 did not work correctly and it did not recognize the p16 protein. We sent you the jpeg files of our Western blots to better explain the problem and Carolyn proposed us to replace the vial with a new one. So, we stopped our experiments and, after a month, we received the new vial of Ab16123 (November, 16th). We immediately noticed that the vial belongs to the same Lot No of the previous one, but to be completely sure we decided to test the antibody. The result was quite disarming: the new antibody doesn’t work! Again, exactly as the first one… Considering that this disappointing situation had already caused a significant delay in our experiment schedule, we contacted the Italian distributor Euroclone, but they just closed the commercial partnership with Abcam and we are now without a national contact. We already received and sent back your feedback questionnaire about Ab16123, but we are writing you to receive an answer as soon as possible, since we strongly need an anti-p16 antibody. So, we would be very happy to have a new free of charge vial of a DIFFERENT Lot No of Ab16123. If this could not be possible, a second option could be to receive a vial of another anti-p16 mouse monoclonal antibody, for instance the Ab16880 or a similar one. We actually do not know alternative solutions. We do really hope to obtain a positive feedback from you. Thank you in advance for your attention.

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Answer

Thank you for your email. I am very sorry to hear that the replacement antibody Carolyn sent you also did not work. I certainly understand that your time is valuable and I would be happy to send you another anti-p16 antibody. I checked our current stock and we still have the same lot of ab16123 available. So instead, as you suggest, I could send you a vial of ab16880. Since you have received products in the past via our Italian distributor Euroclone who we no longer use, I will just need your shipping address so we can ship the antibody directly to you. Again, I apologize that these 2 vials of ab16123 have not performed as expected. I look forward to hearing from you so that we can get the replacement to you as soon as possible.

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Question

LOT NUMBER GR41994-1 ORDER NUMBER 39180 DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE lanes 1,2, 3 and 4: human lung cancer cell line (Calu6)whole cell lysate, lanes 5 and 6: normal mammary epithelial cells (MCF10A)whole cell lysate, PRIMARY ANTIBODY anti-TIMP1 (ab61224) diluted 1/200 in PBS-Tween 0,1%, incubation ON at 4 celcius washed 4 times 5 min with PBS-Tw 0,1% DETECTION METHOD Enhanced Luminol (Perkin Elmer) film X-OMAT exposed 2 and 30 min POSITIVE AND NEGATIVE CONTROLS USED We consider MCF10A cells as positive control ANTIBODY STORAGE CONDITIONS aliquot the same day it arrived at the lab and stored at -20 SAMPLE PREPARATION lanes 1,2, 3 and 4 lysing buffer: HEPES 25mM pH7,5, NaCl 150mM, MgCl2 10mM, EDTA 1mM, Trition 0,1%, Glycerol 10%, aprotinin 10microM and sodium vanadate 1mM lanes 5 and 6 lysing buffer:sodium phosphate 20mM, NaCl 150mM, Triton 1%, EDTA 5mM, PMSF 0,2mg/ml, Aprotinin 10microg/ml, leupeptin 10microg/ml, sodium vanadate 0,25mg/ml Sample buffer 2X: Tris 150mM pH 6,8, SDS 1,2%, Glycerol 30%, beta-mercaptoethanol 15%, bromophenol blue 0,0018% Protocole: samples were mixed with sample buffer 2X (1:1) and boiled for 10 min AMOUNT OF PROTEIN LOADED lanes 1,3 and 5 70microg, lanes 2,4 and 6 20microg ELECTROPHORESIS/GEL CONDITIONS 12% resolving gel for denaturing SDS-PAGE and 4% stacking gel TRANSFER AND BLOCKING CONDITIONS wet transfer with Tris-Glycine-SDS buffer with 20% methanol, 900mA for 90min Blocking: PBS-Tween 0,1%- milk 5%, 90min, RT SECONDARY ANTIBODY anti-Rabbit-HRP (Santa Cruz sc-2004) 1/10000 in PBS-Tw 0,1% 90min washed 4 times 5 min with PBS-Tw 0,1% HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? We changed sample loading buffer. The one before contained less beta-mercaptoethanol and hight molecular weigth appeared on film ADDITIONAL NOTES Is it possible that we detect dimers of TIMP1

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that antibodies ab16123, ab39184 and ab61224 are not providing satisfactory results. The details provided will enable us to investigate these cases and will provide us with vital information for monitoring product quality. Having reviewed these 3 cases, I would like to offer some suggestions to help optimize the results from ab16123, ab39184 and ab61224 : To reduce the background, I recommend to load a maximum of 20µg of samples on the gel. I would suggest to use each primary antibody at a dilution 1/1000 and to dilute each one in the blocking solution. The dilution factor can then be optimized around this value. I would also recommend to try an incubation at room temperature for 2 or 3 hours. Some antibodies bind stonger and more specifically at room temperature. In order to determine the origin of the background I would recommend to run a no primary control in order to see if the background is due to some non-specific binding by the secondary antibody. Last but not least, when testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg . Should the suggestions not improve the results, please do let me know. In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for contacting us. We have indeed in our catalog 5 unconjugated anti-interferon alpha 1 antibodies tested and guaranteed to work in Western Blot and Human samples. Please note that, unfortunately, import restrictions make the goat polyclonal antibody ab86316 unavailable to Canadian customers. The remaining 4 products are very similar. However, I would recommend : - the anti-Interferon alpha antibody clone [AE3] reference ab10077 (www.abcam.com/ab10077) or - the anti-Interferon alpha antibody clone [C10F5] reference ab8317 (www.abcam.com/ab8317). These two mouse monoclonal antibodies, protein A purified, are currently in stock, and their unit size is 250µg which makes them cheaper than the other anti-IFNA1. About the other targets, I agree with you, I would recommend - ab39184 for the detection of TIMP3, - ab61224 for TIMP1, - ab95451 (Mouse monoclonal) for Maspin. ab22354 (Rabbit polyclonal) is also a good choice, - ab16123 for CDKN2A/p16INK4a. ab54210 is also well characterised with 2 publications and 3 Abreviews (customer feedbacks). I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Application
Western blot
Sample
Cow Cell lysate - whole cell (vascular endothelial cells)
Loading amount
20 µg
Specification
vascular endothelial cells
Gel Running Conditions
Reduced Denaturing (15%)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 22 2011

Question
Answer

Thank you for your enquiry. I am sorry it has taken a little time to obtain this information for you. Thank you for your patience. I can confirm that p16 is widely expressed. This antibody has been used successfully in western blotting with HeLa, Saos-2 and SHP-77 whole cell lysates. For tissue sections we would recommend to use bladder tissue. A suitable negative control would be U2OS cells.

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1-10 of 12 Abreviews or Q&A

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