Anti-Cyclin D1 antibody [SP4]

20 product images

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  Image kindly supplied by Dr Karin Birkenkamp-Demtroeder through customer review

  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Gel run under denaturing conditions 4-12% gradient.
  

  This image was generated from the hybridoma version.

  Lane 1: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/200 dilution

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

  All lanes: Whole cell lysate prepared from T24 bladder cancer cells at 25 µg

  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: 34 kDa

  Observed band size: 33 kDa

  Exposure time: 10min

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Lanes 1- 2: Merged signal (red and green). Green - ab16663 observed at 36 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.
  

  ab16663 was shown to react with CCND1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human CCND1 (Cyclin D1) knockout HeLa cell line ab255348 (knockout cell lysate Human CCND1 (Cyclin D1) knockout HeLa cell lysate ab263808) was used. Wild-type HeLa and CCND1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab16663 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/200 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: CCND1 knockout HeLa cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 34 kDa

  Observed band size: 36 kDa

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  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.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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.
  

  This image was generated from the hybridoma version.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  IHC image of ab16663 staining Cyclin D1 in a section of formalin-fixed paraffin-embedded normal human tonsil* performed on a Leica BOND^TM 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 20mins. 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. The inset secondary-only control image is taken from an identical assay without primary antibody.
  

  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.

  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  This image was generated from the hybridoma version.

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (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.
  

  This image was generated from the hybridoma version.

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Lanes 1- 2: Merged signal (red and green). Green - ab16663 observed at 36 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) observed at 50 kDa.
  

  ab16663 was shown to react with Cyclin D1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human CCND1 (Cyclin D1) knockout HeLa cell line ab261760 (knockout cell lysate Human CCND1 (Cyclin D1) knockout HeLa cell lysate ab256864) was used. Wild-type HeLa and CCND1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab16663 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) overnight at 4°C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/200 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: CCND1 knockout HeLa cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 34 kDa

  Observed band size: 36 kDa

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  All lanes: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/1000 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate at 10 µg

  Lane 2: MCF7 (Human breast adenocarcinoma epithelial cell) cell lysate at 10 µg

  Lane 3: CCND1 KO HAP1 cell lysate at 10 µg

  Lane 4: HAP1 (Human chronic myelogenous leukemia near-haploid cell line) cell lysate at 10 µg

  Lane 5: Neuro-2a (Mouse neuroblastoma neuroblast) cell lysate at 10 µg

  Lane 6: NIH/3T3 (Mouse embryonic fibroblast) cell lysate at 10 µg

  Lane 7: C6 (Rat glial tumor glial cell) cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 34 kDa

  Observed band size: 33 kDa

  Exposure time: 2s

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab16663)

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

  This image was generated from the hybridoma version.

  All lanes: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: CCND1 (Cyclin D1) knockout HAP1 whole cell lysate at 20 µg

  Lane 3: A431 whole cell lysate at 20 µg

  Predicted band size: 34 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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.
  

  This image was generated from the hybridoma version.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (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.
  

  This image was generated from the hybridoma version.

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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).
  

  This image was generated from the hybridoma version.

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  Flow Cytometry (Intracellular) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Intracellular 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black). Unlableled control - Unlabelled cells (blue). This image was generated from the hybridoma version.

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  Flow Cytometry (Intracellular) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Intracellular 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black). Unlableled control - Unlabelled cells (blue). This image was generated from the hybridoma version.

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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).

  This image was generated from the hybridoma version.

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  This image was generated from the hybridoma version.

  All lanes: Anti-Cyclin D1 antibody [SP4] (ab137875) at 1/5000 dilution

  All lanes: MCF-7 cell lysate

  Predicted band size: 34 kDa

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  This image was generated from the hybridoma version.

  All lanes: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/25 dilution

  All lanes: MCF7 cell lysate

  Predicted band size: 34 kDa

  Observed band size: 36 kDa

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  This image is courtesy of a customer review submitted by Karin Birkenkamp-Demtroeder

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  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.
  

  This image was generated from the hybridoma version.

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  Flow Cytometry (Intracellular) - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Intracellular 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black). Unlableled control - Unlabelled cells (blue). This image was generated from the hybridoma version.

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  Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663)

  False colour image of Western blot: Anti-Cyclin D1 antibody [SP4] staining at 1/25 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab16663 was shown to bind specifically to Cyclin D1. A band was observed at 35 kDa in wild-type A549 cell lysates with no signal observed at this size in ccnd1 knockout cell line Human CCND1 knockout A549 cell line ab286759 (knockout cell lysate ab300213). To generate this image, wild-type and ccnd1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-Cyclin D1 antibody [SP4] (ab16663) at 1/25 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 3: SH-SY5Y cell lysate at 20 µg

  Lane 4: K562 cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 34 kDa

  Observed band size: 35 kDa

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  Immunohistochemistry - Anti-Cyclin D1 antibody [SP4] (ab16663)

  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CyclinD1 with ab16663 at a dilution of 1/200.

  The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5. ab16663 CyclinD1 was incubated at 37°C for 16min.

  Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)