All lanes : Anti-Geminin antibody [EPR14637] (ab195047) at 1/500 dilution

Lane 1 : Wild-type HeLa lysate
Lane 2 : Geminin DNA Replication Inhibitor knockout HeLa lysate
Lane 3 : HepG2 lysate
Lane 4 : U-87 MG lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 24 kDa

Lanes 1-4: Merged signal (red and green). Green - ab195047 observed at 32 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab195047 Anti-Geminin antibody [EPR14637] was shown to specifically react with Geminin DNA Replication Inhibitor in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264972 (knockout cell lysate ab257450) was used. Wild-type and Geminin DNA Replication Inhibitor knockout samples were subjected to SDS-PAGE. ab195047 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 500 and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF-7 (Human breast adenocarcinoma cell line) cells labeling Geminin with ab195047 at 1/100 followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500(green). Confocal image showing nuclear staining on MCF-7 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500  (red).

The negative controls are as follows:-
-ve control 1 - ab195047 at 1/100 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500.

All lanes : Anti-Geminin antibody [EPR14637] (ab195047) at 1/500 dilution

Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) treated with thymidine whole cell lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) treated with nocodazole whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 24 kDa


Exposure time: 3 minutes

Blocking buffer and diluting buffer was concentration 5% NFDM /TBST

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Geminin with ab195047 at 1/500 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500. Nucleus staining on Human tonsil tissue is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody and ab97051 at 1/500 as secondary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human triple-negative breast cancer tissue labeling Geminin with ab195047 at 1/500 followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500. Nucleus staining on Human triple-negative breast cancer tissue is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody and ab97051 at 1/500 as secondary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab195047 staining Geminin in the HeLa cell line from Human cervix by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 Samples were incubated with primary antibody (1/50 in PBS ) for 1 hour at 22°C. Ab150081 (1/200) was used as the secondary antibody.

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