Anti-RPA32/RPA2 antibody [EPR2877Y] - Mouse IgG1 (Chimeric)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RPA32/RPA2 antibody [EPR2877Y] - Mouse IgG1 (Chimeric) (AB324313)

ab324313 staining RPA2 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab324313 at 5µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min).Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPA32/RPA2 antibody [EPR2877Y] - Mouse IgG1 (Chimeric) (AB324313)

Immunofluorescence staining of RPA2 in sections of formalin-fixed paraffin-embedded normal human colon.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate (pH6.0) for 20 minutes. The section was then incubated at room temperature for 1 hour with ab324313 at 1/500 dilution and then incubated for 1 hour with ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• WB

Lab

Western blot - Anti-RPA32/RPA2 antibody [EPR2877Y] - Mouse IgG1 (Chimeric) (AB324313)

False colour image of Western blot : Anti-RPA32/RPA2 antibody [EPR2877Y] - Mouse IgG1 (Chimeric) (ab324313) staining at 1/1000 dilution, shown in black; Rabbit anti-alpha tubulin loading control staining, shown in magenta. In Western blot, ab324313 was shown to bind specifically to RPA2 protein in HeLa whole cell lysate. HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 20ug. Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat Anti-Mouse IgG Fc (HRP) preadsorbed (ab98717) at 1 : 10000 and Goat anti-Rabbit IgG H&L 680RD at 1 : 20000.

All lanes:

Western blot - Anti-RPA32/RPA2 antibody [EPR2877Y] - Mouse IgG1 (Chimeric) (ab324313) at 1/1000 dilution

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lane 1:

Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-preadsorbed-ab98717'>ab98717</a>) at 1/10000 dilution

Lane 1:

Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 29 kDa

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