Anti-Dnmt1 antibody (ab19905)

  Western blot - Dnmt1 antibody (ab19905)

All lanes : Anti-Dnmt1 antibody (ab19905) at 1 µg/ml

Lane 1 : HeLa nuclear lysate
Lane 2 : HeLa nuclear lysate with Dnmt1 peptide (ab21999) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 183 kDa
Observed band size : 183 kDa


ab19905 specifically recognises Dnmt1 at 183 kDa in HeLa nuclear extracts (lane1), which is efficiently blocked using the immunising peptide (ab21999) (lane2).

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  Immunoprecipitation - Anti-Dnmt1 antibody (ab19905)

Dnmt1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Dnmt1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19905.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 183kDa: Dnmt1.

  Immunocytochemistry/ Immunofluorescence - Dnmt1 antibody (ab19905)

ab19905 at a 1/200 dilution staining paraformaldehyde fixed asynchronous HeLa cells by ICC/IF. The antibody was incubated with the cells for 30 minutes and then bound antibody was detected using a Cy3 conjugated goat anti-rabbit antibody. Nuclei were visualised using DAPI staining. Ab19905 gives a pattern that is predominantly enriched within nuclei.

This image is courtesy of an Abreview submitted by Kirk McManus.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Dnmt1 antibody (ab19905)

Staining of human tonsil tissue was performed using ab19905. Strong nuclear staining was observed in germinal center cells and scattered cells in the interfollicular area. T cells were weakly to moderately positive and endothelial cells were positive.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Dnmt1 antibody (ab19905)

ab19905 (1/100) staining Dnmt1 in paraffin-embbeded mouse testis tissue sections. Tissue underwent fixation in formaldehyde, heat-mediated antigen retrieval in 10mM citrate buffer pH 6.0 and blocking (30 minutes, 8% milk). A biotinylated anti-rabbit IgG (H+L) antibody was used as the secondary. For further experimental details please refer to abreview.

This image is courtesy of an anonymous Abreview

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  Western blot - Dnmt1 antibody (ab19905)

Anti-Dnmt1 antibody (ab19905) at 1 µg/ml + IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 183 kDa
Observed band size : 183 kDa
Additional bands at : 33 kDa,38 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 3 minutes

  Immunocytochemistry/ Immunofluorescence - Dnmt1 antibody (ab19905)

ICC/IF image of ab19905 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19905, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 1% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml.