Anti-DDB2 antibody [2246C4a]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDB2 antibody [2246C4a] (AB51017)

IHC image of ab51017 staining in human normal cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab51017, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DDB2 antibody [2246C4a] (AB51017)

ICC/IF image of ab51017 stained HeLa cells. The cells were 4% formaldehyde 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 (ab51017, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.

• Flow Cyt

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Flow Cytometry - Anti-DDB2 antibody [2246C4a] (AB51017)

Overlay histogram showing HeLa cells stained with ab51017 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51017, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed.

• WB

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Western blot - Anti-DDB2 antibody [2246C4a] (AB51017)

All lanes:

Western blot - Anti-DDB2 antibody [2246C4a] (ab51017) at 1/50 dilution

All lanes:

HeLa whole cell lysate at 50 µg

Secondary

All lanes:

Mouse IgG antibody at 1/2500 dilution

Predicted band size: 48 kDa

Observed band size: 45 kDa

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• WB

CiteAb

Western blot - Anti-DDB2 antibody [2246C4a] (AB51017)

DDB2 western blot using anti-DDB2 antibody [2246C4a] ab51017. Publication image and figure legend from Luch, A., Frey, F. C., et al., 2014, PLoS One, PubMed 24722772.

ab51017 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 ab51017 please see the product overview.

Association of UV-DDB with damaged chromatin.(A) Flow diagram illustrating how chromatin was dissected to monitor the binding of UV-DDB. Unbound proteins were released by salt (0.3 M NaCl) extraction and the remaining chromatin was solubilized by MNase digestion. (B) Western blot visualization of the chromatin partitioning of UV-DDB using antibodies against DDB2. GAPDH (glyceraldehyde 3-phosphate dehydrogenase), marker of unbound proteins; histone H3, marker of chromatin. Human fibroblasts were exposed for 18 h to formaldehyde or UV-irradiated at the indicated doses. (C) Quantification of three independent binding assays demonstrating the differential interaction of DDB2 with formaldehyde- and UV-damaged chromatin (error bars, S.D.). (D) Release of chromatin-bound DDB2 and histone H3 by high-salt extraction. After incubation with 0.3 M NaCl buffer, the chromatin was dissolved with 2.5 M NaCl, thus liberating non-covalently bound chromatin proteins.

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