Anti-Cyclin D1 antibody [EPR2241] - C-terminal

• WB

Lab

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Western blot : Anti-CCND1 antibody [EPR2241] (ab134175) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab134175 was shown to bind specifically to CCND1. A band was observed at 34 kDa in wild-type A549 cell lysates with no signal observed at this size in CCND1 knockout cell line. 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 [EPR2241] - C-terminal (ab134175) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

CCND1 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CCND1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-ccnd1-knockout-a549-cell-line-ab286759'>ab286759</a>)

Lane 3:

SH-SY5Y cell lysate at 20 µg

Lane 4:

K562 cell lysate at 20 µg

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Immunofluorescent staining of Ramos (Human Burkitt's lymphoma cell line) cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab134175 at a dilution of 1/50. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counterstained with DAPI.

The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

IHC image of ab134175 staining Cyclin D1 in human 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 ab134175, 5μg/ml working concentration, 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 hematoxylin 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Immunohistochemical staining of paraffin embedded human endometrial adenocarcinoma with purified ab134175 at a dilution of 1/100. An HRP goat anti-rabbit (ab97051) was used as the secondary antibody at a dilution of 1/500 and the sample was counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (inset).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Unpurified ab134175 staining Cyclin D1 in MCF7 (Human breast adenocarcinoma cell line) cells treated with KN-93 (ab120980).

The cells were fixed with 100% methanol (5min) 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 ab134175 at 10μg/ml and ab7291 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an Goat anti-Rabbit Alexa 488 secondary (ab150081) at 2 μg/ml (shown in green) and Goat anti-Mouse Alexa 594 secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls : 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Immunohistochemistry was performed on human head and neck squamous cell carcinoma tissue using a rabbit monoclonal antibody against the CCND1 protein ab134175 on 3-μm slides using 224 paraffin sections via the standard SP method.

Panel C : An example of low Cyclin D1 expression.

Panel G : An example of high Cyclin D1 expression.

For full image please see paper.

Feng et al PLoS One. 2011;6(10):e26399. doi: 10.1371/journal.pone.0026399. Epub 2011 Oct 31. Fig 1. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• ICC/IF

AbReview40418****

Immunocytochemistry/ Immunofluorescence - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Immunocytochemical analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, labeling Cyclin D1 with ab134175 at a dilution of 1/200. Cells were paraformaldehyde fixed and permeabilized with 0.5% Triton X-100 in PBS. Incubation with the primary antibody was for 1 hour at 22°C. Cells were counterstained with DAPI following immunostaining.

This image is courtesy of an Abreview submitted by Kirk Mcmanus.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Immunohistochemical analysis of paraffin-embedded human mantle cell lymphoma tissue, labeling Cyclin D1 with unpurified ab134175 at 1/100 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling Cyclin D1 with ab134175 at a concentration of 1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

Anti-Cyclin D1 antibody [EPR2241] ab134175 was incubated for 16mins at 37°C. Sections were counterstained 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).

• IP

Lab

Immunoprecipitation - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

ab134175 (purified) at 1/30 immunoprecipitating cyclin D1 in A431 (Human epidermoid carcinoma cell line) cells. For western blotting, an HRP-conjugated goat anti-rabbit IgG, was used as the secondary antibody (1/1000).

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175)

Predicted band size: 34 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

IHC image of ab134175 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 ab134175, 5μg/ml working concentration, 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 hematoxylin 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.

• WB

Lab

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 1/100000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma cell line) cell lysate at 20 µg

Lane 2:

LNCap (Human prostate cancer cell line) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

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• WB

Unknown

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

MG-63 cells were incubated at 37°C for 24h with vehicle control (0 μM) and different concentrations of 3-aminobenzamide (3-AB) (ab141069). Decreased expression of cyclin D1 (unpurified ab134175) in MG-63 cells correlates with an increase in 3-aminobenzamide (3-AB) concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with unpurified ab134175 at 1/500 dilution and ab8226 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody (ab97051) at 1/10000 dilution and visualised using ECL development solution.

All lanes:

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175)

Predicted band size: 34 kDa

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Exposure time: 3min

• WB

Lab

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 1/20000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma cell line) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

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• WB

Lab

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 1/10000 dilution

Lane 1:

Mouse kidney at 20 µg

Lane 2:

Mouse spleen at 20 µg

Lane 3:

Rat heart at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

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• WB

Lab

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

Blocking and dilution buffer : 5% NFDM/TBST.

Lane 1:

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 0.04 µg/mL

Lane 2:

Western blot - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 2 µg/mL

Lane 1:

His-Tagged full-length human Cyclin D1 (aa 1 to 295) recombinant protein at 0.015 µg

Lane 2:

His-Tagged full-length human Cyclin D2 (aa 1 to 289) recombinant protein at 0.015 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 34 kDa

Observed band size: 37 kDa

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Exposure time: 3min

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Cyclin D1 antibody [EPR2241] - C-terminal (AB134175)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.