Overview

  • Product name
    Anti-Cyclin D1 antibody [SP4]
    See all Cyclin D1 primary antibodies
  • Description
    Rabbit monoclonal [SP4] to Cyclin D1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC, ICC/IF, IHC-P, WB, IHC-P, IHC-Fr, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Cyclin D1 (C terminal).

  • Epitope
    C-terminus
  • Positive control
    • Breast carcinomas, mantle cell lymphoma, MCF7 cell lysate IHC: Rat Esophagus (FFPE) ICC/IF: MCF7 cells, C6, Neuro-2a and HAP1 cells (HAP1-CCND1 knockout cells used as negative cell line) FC: MCF-7, NIH/3T3 and C6 cells

Properties

Applications

Our Abpromise guarantee covers the use of ab16663 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 Use at an assay dependent concentration.
ICC/IF 1/50 - 1/250.
IHC-P 1/100.
WB 1/25 - 1/200. Detects a band of approximately 36 kDa (predicted molecular weight: 33 kDa).
IHC-P 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Deparaffinization: Deparaffinize slides using xylene or xylene alternative and graded alcohols.

Antigen Retrieval: Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min.

Primary Antibody Incubation: Incubate for 30 minutes at room temperature.

Slide Washing: Slides must be washed in between steps. Rinse slides with PBS/0.05% Tween.

IHC-Fr Use at an assay dependent concentration.
Flow Cyt 1/30.

Target

  • Function
    Essential for the control of the cell cycle at the G1/S (start) transition.
  • Involvement in disease
    Note=A chromosomal aberration involving CCND1 may be a cause of B-lymphocytic malignancy, particularly mantle-cell lymphoma (MCL). Translocation t(11;14)(q13;q32) with immunoglobulin gene regions. Activation of CCND1 may be oncogenic by directly altering progression through the cell cycle.
    Note=A chromosomal aberration involving CCND1 may be a cause of parathyroid adenomas. Translocation t(11;11)(q13;p15) with the parathyroid hormone (PTH) enhancer.
    Defects in CCND1 are a cause of multiple myeloma (MM) [MIM:254500]. MM is a malignant tumor of plasma cells usually arising in the bone marrow and characterized by diffuse involvement of the skeletal system, hyperglobulinemia, Bence-Jones proteinuria and anemia. Complications of multiple myeloma are bone pain, hypercalcemia, renal failure and spinal cord compression. The aberrant antibodies that are produced lead to impaired humoral immunity and patients have a high prevalence of infection. Amyloidosis may develop in some patients. Multiple myeloma is part of a spectrum of diseases ranging from monoclonal gammopathy of unknown significance (MGUS) to plasma cell leukemia. Note=A chromosomal aberration involving CCND1 is found in multiple myeloma. Translocation t(11;14)(q13;q32) with the IgH locus.
  • Sequence similarities
    Belongs to the cyclin family. Cyclin D subfamily.
  • Post-translational
    modifications
    Phosphorylation at Thr-286 by MAP kinases is required for ubiquitination and degradation following DNA damage. It probably plays an essential role for recognition by the FBXO31 component of SCF (SKP1-cullin-F-box) protein ligase complex.
    Ubiquitinated, primarily as 'Lys-48'-linked polyubiquitination. Ubiquitinated by a SCF (SKP1-CUL1-F-box protein) ubiquitin-protein ligase complex containing FBXO4 and CRYAB (By similarity). Following DNA damage it is ubiquitinated by some SCF (SKP1-cullin-F-box) protein ligase complex containing FBXO31. Ubiquitination leads to its degradation and G1 arrest. Deubiquitinated by USP2; leading to stabilize it.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • AI327039 antibody
    • B cell CLL/lymphoma 1 antibody
    • B cell leukemia 1 antibody
    • B cell lymphoma 1 protein antibody
    • B-cell lymphoma 1 protein antibody
    • BCL 1 antibody
    • BCL-1 antibody
    • BCL-1 oncogene antibody
    • BCL1 antibody
    • BCL1 oncogene antibody
    • ccnd1 antibody
    • CCND1/FSTL3 fusion gene, included antibody
    • CCND1/IGHG1 fusion gene, included antibody
    • CCND1/IGLC1 fusion gene, included antibody
    • CCND1/PTH fusion gene, included antibody
    • CCND1_HUMAN antibody
    • cD1 antibody
    • Cyl 1 antibody
    • D11S287E antibody
    • G1/S specific cyclin D1 antibody
    • G1/S-specific cyclin-D1 antibody
    • Parathyroid adenomatosis 1 antibody
    • PRAD1 antibody
    • PRAD1 oncogene antibody
    • U21B31 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: CCND1 (Cyclin D1) knockout HAP1 whole cell lysate (20 µg)
    Lane 3: A431 whole cell lysate (20 µg)

    Lanes 1 - 3: Merged signal (red and green). Green - ab16663 observed at 34 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab16663 was shown to specifically recognize CCND1 (Cyclin D1) in wild-type HAP1 cells as signal was lost at the expected MW in CCND1 (Cyclin D1) knockout cells. Wild-type and CCND1 (Cyclin D1) knockout samples were subjected to SDS-PAGE. Ab16663 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/200 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence analysis of C6 (rat glial tumor glial cell) cells labeling Cyclin D1 with purified ab16663 at 1/50 (5.42µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • IHC image of ab16663 staining Cyclin D1 in rat esophagus formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab16663, 1:100 dilution, 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. No primary antibody was used in the secondary only control (shown on the inset).
    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.

  • Immunocytochemistry/ Immunofluorescence analysis of Neuro-2a (mouse neuroblastoma neuroblast) cells labeling Cyclin D1µ with purified ab16663 at 1/50 (5.42µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • ab16663 staining Cyclin D1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16663 at a working dilution of 1/250 and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-rabbit AlexaFluor® 488 (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Flow cytometry analysis of MCF-7 (human breast carcinoma) labeling Cyclin D1 with purified ab16663 at 1/30 dilution (9.03 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlableled control - Unlabelled cells (blue).

  • Flow cytometry analysis of C6 (rat glioma) labeling Cyclin D1 with purified ab16663 at 1/30 dilution (9.03 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlableled control -  Unlabelled cells (blue).

  • Flow cytometry analysis of NIH/3T3 (mouse embryo) labeling Cyclin D1 with purified ab16663 at 1/30 dilution (9.03 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlableled control - Unlabelled cells (blue).

  • ab16663 staining Cyclin D1 in wild-type HAP1 cells (top panel) and CCND1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16663 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Immunohistochemical analysis of mouse testis tissue, staining Cyclin D1 with ab16663.

    Antigen retrieval was performed via Tris-EDTA buffer. Sections were blocked with 3% BSA and incubated with primary antibody (1/50) overnight at 4°C. An AlexaFluor®594-conjugated secondary antibody was used to detect staining.
  • Anti-Cyclin D1 antibody [SP4] (ab137875) at 1/5000 dilution + MCF-7 cell lysate

    Predicted band size: 33 kDa

  • Lane 1 : Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/200 dilution
    Lane 2 : Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/400 dilution

    All lanes : Whole cell lysate prepared from T24 bladder cancer cells

    Lysates/proteins at 25 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit IgG conjugated to HRP at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 33 kDa
    Observed band size: 33 kDa


    Exposure time: 10 minutes


    Gel run under denaturing conditions 4-12% gradient.

    See Abreview

  • Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/25 dilution + MCF7 cell lysate

    Predicted band size: 33 kDa
    Observed band size: 36 kDa
    why is the actual band size different from the predicted?

  • ab16663 staining Cyclin D1 in Human urinary tract tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% BSA for 30 minutes at room temperature; antigen retrieval was by heat mediation in citrate buffer. Samples were incubated with primary antibody (1/100 in PBS) for 1 hour. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

    See Abreview

  • Human mantle cell lymphoma stained with ab16663.

References

This product has been referenced in:
  • Gu W  et al. Dnd1-mediated epigenetic control of teratoma formation in mouse. Biol Open 7:N/A (2018). Read more (PubMed: 29378702) »
  • Li Y  et al. The anti-tumor effects of Mfn2 in breast cancer are dependent on promoter DNA methylation, the P21Ras motif and PKA phosphorylation site. Oncol Lett 15:8011-8018 (2018). Read more (PubMed: 29731912) »
See all 91 Publications for this product

Customer reviews and Q&As

1-5 of 5 Q&A

Answer

ab16663 is serum free. I hope this is helpful. Please do not hesitate to contact me with any additional questions.

Read More

Question
Answer

Thank you for your telephone enquiry yesterday regarding antibody ab16663. I can confirm that the concentration of this antibody is around 0.1mg/ml. However,as discussed on the phone, the antibody is supplied as cell culture supernatant which means the concentration will vary slightly and this is an approximate value. I hope this information is helpful. Should you have any further questions, please do not hesitate to contact us again.

Read More

Answer

Thank you for your enquiry. The homology of the peptide used to generate ab16663 shows ~70% homology with both cyclin D2 and D3. There is a possibility of cross-reaction, but we do not have data to confirm it. Please let me know if you have additional questions.

Read More

Answer

Thank you for your enquiry. Ab16663 is a rabbit IgG antibody, so you should use an anti-rabbit IgG secondary antibody. At the bottom of the online datasheet there is also a list of compatible secondaries which can be used. If you have any additional questions, please contact us again.

Read More

Question


Issue: No bands on the western blots with two different Abcam antibodies
Samples: cell lysates from Jurkat and U937 cell lines, as well as from
primary human PBMC cells (isolated from total blood by gradient
centrifugation). We tried using 20 and 40 ug of total protein/lane.
Sample buffer: we tried the following - RIPA (Cell Signaling), NuPAGE
LDS sample buffer (Invitrogen), M-Per (Pierce), always supplemented
with protease and phosphatase inhibitor cocktails (Roche)
Acrylamide gels: 10% cast, or 12% pre-cast from Bio-Rad, run at 100V
constant for 50 min and transferred at 300 mA constant for 60 min
using methanol transfer buffer and 0.2 um nitrocellulose membranes.
Blocking in 5% non-fat dry milk + 0.5% Tween-20 in TBS for 1 h (RT).
Primary antibodies: ab89911 (anti-RAGE, lot# GR54357-1 and GR-54357-4)
and ab16663 (anti-Cyclin D1, lot# GR 62215-1)
Concentration of primary antibodies: RAGE - 1:500 dilution; Cyclin D1
- 1:200 dilution in PBS (incubation O/N @ 4C)
Secondary antibodies:
anti-mouse (RAGE), anti-rabbit (Cyclin D1)-HRP conjugated primary
antibodies (Cell Signaling, 1:1000 dilution in TBS, for both -
incubation for 1 h @ RT)
ECL:
Supersignal West Pico Chemiluminescent Substrate, Pierce Technology
Imaging:
Bio-Rad Gel-Doc Xr
Please note that in the same membranes (cut out at different molecular
weight heights) we were able to detect other proteins (p53, p62,
GAPDH) without any problems, suggesting that the samples and sample
preparation, western protocol (run/transfer), secondary antibodies and
ECL are OK. We also have mRNA data from these same cells indicating
that they do express RAGE at the mRNA level. We have also checked the
membranes with Ponceau solution to verify transfer. Also the protein
ladder standards were always adequately separated and transferred.

Read More
Answer

I am sorry to hear that these antibodies are not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the resuts. I would also appreciate if you can confirm some further details:
1) How long is your incubation? Overnight incubation at 4 degrees allows for the strongest images.

2) Do your sample types express high amounts of these targets?

3) For cyline D1, although we recommend using 1/200, there is an image on the datasheet with a much stronger band using the primary antibody at 1/25. I would suggest increasing the antibody dilutions even further in both cases, since as you state it is not the sample preparation that is the problem but rather getting a good signal from the antibody.
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.

Read More

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up