All lanes : Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 1/100000 dilution (purified)

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) cell lysate
Lane 2 : LNCap (Human prostate cancer cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa
Observed band size: 34 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.

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).

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.

 

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.

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 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

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.

All lanes : Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 1/20000 dilution (purified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : A431 (Human epidermoid carcinoma cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa
Observed band size: 34 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 1/10000 dilution (purified)

Lane 1 : Mouse kidney
Lane 2 : Mouse spleen
Lane 3 : Rat heart

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 34 kDa
Observed band size: 34 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Lane 1 : Anti-Cyclin D1 antibody [EPR2241] - C-terminal (ab134175) at 0.04 µg/ml
Lane 2 : 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
Lane 2 : His-Tagged full-length human Cyclin D2 (aa 1 to 289) recombinant protein

Lysates/proteins at 0.015 µg per lane.

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

Predicted band size: 34 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

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.

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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.

Equilibrium disassociation constant (K_D)
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