All lanes : Anti-Geminin antibody [EPR23754-87] (ab246509) at 1/1000 dilution

Lane 1 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 24 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 18798731).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Geminin with ab246509 at 1/100 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil (PMID: 12107111). The section was incubated with ab246509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of 80% methanol fixed, 0.1% Tween-20 permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Geminin with ab246509 at 1/400 dilution (0.1µg) (Right panel) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left panel). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were pre-treated with 20ug/ml RNaseA for 30 mins to minimize the binding between PI and RNA. Then intracellular stained with ab246509 and PI. Increase expression in S phase and G2/M phase was reported in literature (PMID: 28179914).

Geminin was immunoprecipitated from 0.35 mg mouse testis tissue lysate with ab246509 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab246509 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse testis tissue lysate 10µg

Lane 2: ab246509 IP in mouse testis tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab246509 in mouse testis tissue lysate

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

Exposure time: 3 minutes.

 

All lanes : Anti-Geminin antibody [EPR23754-87] (ab246509) at 1/1000 dilution

Lane 1 : Mouse testis tissue lysate
Lane 2 : MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 4 : Rat testis tissue lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 24 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 18798731).

Exposure times. Lane 1, 4: 15 seconds; Lane 2, 3: 37 seconds; Lane 5: 3 minutes.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Geminin with ab246509 at 1/500 followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse testis (PMID: 22898305, PMID: 28690291). The section was incubated with ab246509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse breast carcinoma tissue labeling Geminin with ab246509 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse breast carcinoma (PMID: 12107111). The section was incubated with ab246509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling Geminin with ab246509 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat testis (PMID: 22898305, PMID: 28690291). The section was incubated with ab246509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized MEF (Mouse embryonic fibroblast (immortalized)) cells labelling Geminin with ab246509 at 1/400 dilution (0.1µg) (Right panel) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left panel). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293 (Human embryonic kidney epithelial cell) cells labelling Geminin with ab246509 at 1/400 dilution (0.1µg) (Right panel) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left panel). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.