Anti-CCDC98 antibody [EPR6310(2)]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CCDC98 antibody [EPR6310(2)] (AB139191)

Immunofluorescent analysis of HeLa cells labelling CCDC98 with ab139191 at 1/100 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CCDC98 antibody [EPR6310(2)] (AB139191)

Overlay histogram showing HeLa cells stained with ab139191 (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 followed by the antibody (ab139191 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IP

Lab

Immunoprecipitation - Anti-CCDC98 antibody [EPR6310(2)] (AB139191)

CCDC98 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab139191 at 1/60 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab139191 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab139191 in HeLa whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-CCDC98 antibody [EPR6310(2)] (ab139191)

Predicted band size: 47 kDa

Observed band size: 47 kDa

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• WB

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Western blot - Anti-CCDC98 antibody [EPR6310(2)] (AB139191)

All lanes:

Western blot - Anti-CCDC98 antibody [EPR6310(2)] (ab139191) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

MCF7 cell lysate at 10 µg

Lane 3:

293T (Human embryonic kidney epithelial cell) cell lysate at 10 µg

Lane 4:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 47 kDa

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• WB

CiteAb

Western blot - Anti-CCDC98 antibody [EPR6310(2)] (AB139191)

CCDC98 western blot using anti-CCDC98 antibody [EPR6310(2)] ab139191. Publication image and figure legend from Anantha, R. W., Simhadri, S., et al., 2017, Elife, PubMed 28398198.

ab139191 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 ab139191 please see the product overview.

Sequence alterations generated in BRCA1 and their effects on protein-protein interactions and HR.(A) Domain structure of BRCA1 and the binding sites for its interacting partners. NES, nuclear export signal; NLS, nuclear localization signal. (B) Effects of the BRCA1 variants on the binding of BARD1, PALB2, BRIP1 and Abraxas. The 3xMyc-tagged BRCA1 proteins were transiently expressed in 293T cells and IPed with an anti-Myc antibody. WCE, whole cell extract. (C) Schematic of the HR reporter assay. The DR-GFP reporter contains two defective copies of the GFP gene, one disrupted by an I-SceI site and the other lacking a promoter. I-SceI cutting of the first copy generates a DSB, and repair by HR with the second copy as a template leads to restoration of a functional GFP gene. (D) HR activities of the variants relative to the wt BRCA1 protein. Data shown are the means from two to seven independent experiments for each variant or mutant. Error bars represent standard deviations (SDs). The grey bars indicate variants that are among the top 20 missense variants but are already present in BRCA1 cDNAs obtained from three independent sources. The calculated cutoff threshold is indicated by horizontal lines. **p<0.01. See Figure 1—source data 1 for details. (E) Levels of BRCA1 protein following knockdown and re-expression. Cells treated with a control siRNA (NSC1) were used as a control for the endogenous protein abundance.DOI : http : //dx.doi.org/10.7554/eLife.21350.00310.7554/eLife.21350.004Figure 1—source data 1.HR activities of the BRCA1 variants and mutants analzyed in this study.See texts and materials and methods for details.DOI : http : //dx.doi.org/10.7554/eLife.21350.004HR activities of the BRCA1 variants and mutants analzyed in this study.See texts and materials and methods for details.DOI : http : //dx.doi.org/10.7554/eLife.21350.004Capacity of the BRCA1 variants to bind BARD1, PALB2, CtIP and BRIP1.The 3xMyc-tagged BRCA1 proteins were transiently expressed in 293T cells and IPed with an anti-Myc antibody. Note that the amount of CtIP co-IPed with BRCT mutants R1699Q and A1708E was the same as the background level in the vector lane.DOI : http : //dx.doi.org/10.7554/eLife.21350.005Expression levels of BRCA1 BRCT missense and truncating mutants in U2OS/DR-GFP cells first depleted of the endogenous BRCA1 and then transfected with cDNA expressing the mutants.Cells untreated with any siRNA were used as a control for the endogenous protein abundance (spliced from the same gel).DOI : http : //dx.doi.org/10.7554/eLife.21350.006

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