The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
1/25 - 1/200. Detects a band of approximately 36 kDa (predicted molecular weight: 33 kDa).
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.
FunctionEssential for the control of the cell cycle at the G1/S (start) transition.
Involvement in diseaseNote=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 similaritiesBelongs to the cyclin family. Cyclin D subfamily.
Post-translational modificationsPhosphorylation 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.
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).
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] (ab16663)Image from McIver SC et al., PLoS One. 2012;7(4):e35553. Epub 2012 Apr 20. Fig 7.; doi:10.1371/journal.pone.0035553; April 20, 2012, PLoS ONE 7(4): e35553.
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.
Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] (ab16663)This image is courtesy of an Abreview submitted by Karin Birkenkamp-Demtroeder
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.
References for Anti-Cyclin D1 antibody [SP4] (ab16663)
This product has been referenced in:
Peng W & Feng J Long noncoding RNA LUNAR1 associates with cell proliferation and predicts a poor prognosis in diffuse large B-cell lymphoma. Biomed Pharmacother77:65-71 (2016).
Read more (PubMed: 26796267) »
Kaistha BP et al. PLAC8 Localizes to the Inner Plasma Membrane of Pancreatic Cancer Cells and Regulates Cell Growth and Disease Progression through Critical Cell-Cycle Regulatory Pathways. Cancer Res76:96-107 (2016).
Read more (PubMed: 26669866) »