Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel (AB228528)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Cyclin B1 with ab181593 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasm and weak nuclear staining on HeLa cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows :
1. ab181593 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel (AB228528)

ab124821 staining Cdk6 in wild-type HAP1 cells (top panel) and Cdk6 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 ab124821 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudo colour 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel (AB228528)

ab32147 staining Cdk2 in the HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/200). ab150078 (1/500) an Alexa Fluor^® 555-conjugated Goat anti-rabbit IgG was used as the secondary antibody. Nuclei were counterstained with DAPI.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel (AB228528)

Unpurified ab134175 staining Cyclin D1 in MCF7 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 labelled 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.

• WB

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Western blot - Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel (AB228528)

Lane 1 : Wild-type DLD-1 cell lysate (20 μg)

Lane 2 : Wild-type DLD-1 20 μM 2,3-DCPE for 16hrs treated cell lysate (20 μg)

Lane 3 : p21 knockout DLD-1 cell lysate (20 μg)

Lane 4 : p21 knockout 20 μM 2,3-DCPE for 16hrs DLD-1 cell lysate (20 μg)

Lane 5 : HT1080 cell lysate (20 μg)

Lanes 1 - 5 : Merged signal (red and green). Green - Anti-p21 antibody [EPR3993] (ab109199) observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109199 was shown to recognize p21 in WT DLD-1 cells with 2,3-DCPE treatment along with additional cross-reactive bands. When p21 knockout DLD-1 cells +/- 2,3-DCPE treatment were used, no band was observed. Wild-type and p21 knockout samples were subjected to SDS-PAGE. ab109199 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-p21 antibody [EPR3993] (<a href='/en-us/products/primary-antibodies/p21-antibody-epr3993-ab109199'>ab109199</a>)

Predicted band size: 18 kDa

Observed band size: 20 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Cell Cycle (Cdk2, Cdk6, Cyclin B1, Cyclin D1, p21, p27 KIP 1) Antibody Sampler Panel (AB228528)

ab32034 showing positive staining in Ovarian carcinoma tissue.