Anti-MCM5 antibody [EP2683Y]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MCM5 antibody [EP2683Y] (AB75975)

Immunocytochemistry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling MCM5 with Purified ab75975 at 1 : 50 dilution (2.1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488,ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MCM5 antibody [EP2683Y] (AB75975)

Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling MCM5 with Purified ab75975 at 1/20 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488 ,ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCM5 antibody [EP2683Y] (AB75975)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling MCM5 with purified ab75975 at 1/1000 dilution (0.11 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Unknown

Western blot - Anti-MCM5 antibody [EP2683Y] (AB75975)

Blocking Buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MCM5 antibody [EP2683Y] (ab75975) at 1/1000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

Mouse spleen lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 82 kDa

Observed band size: 82 kDa

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• WB

CiteAb

Western blot - Anti-MCM5 antibody [EP2683Y] (AB75975)

MCM5 western blot using anti-MCM5 antibody [EP2683Y] ab75975. Publication image and figure legend from Shao, Z., Koh, W., et al., 2020, Sci Rep, PubMed 32313136.

ab75975 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab75975 please see the product overview.

Validation of Two-week-old Mouse Heart RNA-Sequencing Results by RT-qPCR and Western Blot. RNA and protein samples analyzed were extracted from WT and Lmna-/- mouse heart tissues at 2 weeks of age. (a) RT-qPCR validation of down-regulated genes (AURKA, BRCA1, CDK1, CHEK1, Cyclin B1, MCM5, PLK1) identified by RNA-Sequencing. The housekeeping gene S18 was used as a control gene for both WT and Lmna-/- samples. ∆∆ cycle threshold (Ct) method was used for RT-qPCR analysis. Average WT expression level was normalized to one and Lmna-/- expression levels were calculated over average WT level and showed as "mean ± SEM" on Y axis. The expression levels of all the listed genes were significantly lower in 2-week Lmna-/- mouse hearts than in 2-week WT mouse hearts (p < 0.01). (b) RT-qPCR validation of up-regulated genes (EPHX1, NDUFA13, NRF2, PLK3, SQSTM1) identified by RNA-Sequencing. The same ∆∆Ct method was used for analysis. The relative expression levels of all the listed genes are showed as "mean ± SEM" on Y axis. The expression levels in 2-week-old Lmna-/- mouse hearts were significantly higher than those in 2-week-old WT mouse hearts for all the genes (p < 0.05). (c) Western blot validation of protein expressions of representative down-regulated (CDK1, Cyclin B1 and MCM5) and up-regulated (NRF2 and PLK3) genes. GAPDH was used as a loading control. Three repeated experiments were conducted for each protein with similar results showed in the figure.

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