Anti-HA tag antibody - ChIP Grade (ab9110)

Immunofluorescent analysis of 4% paraformaldehyde (PFA) fixed, permeablised with 0.1% Triton X-100 CHO cells transfected with GFP-HA constructs (CHO-GFP-HA) labelling HA tag with ab9110 at 5µg/ml, followed by Donkey Anti-Rabbit IgG(H&L), Alexa 594 conjugated antibody at 2.5µg/ml (Red). Nucleus was counterstained with DAPI (Blue). Parallel staining in parental CHO cell line as negative control.

ab9110 staining HA-tagged proteins in HeLa cells by ICC/IF (Immunocytochemistry/immunoflurescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% saponin and blocked with 3% serum for 30 minutes at 37°C. Samples were incubated with primary antibody (2 µg/ml) in 1x PBS for 1 hour at 37°C. An Alexa Fluor^® 488-conjugated Goat polyclonal to rabbit was used as secondary antibody. 

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ab9110 was diluted to 4 µg/mg lysate and incubated with a nuclear lysate of HEK293T cells transiently expressing HA-tagged protein and a Protein A matrix for 2 hours a 23°C to achieve immunoprecipitation.  1000 µg of lysate was present in the input. 

A HRP-conjugated anti-rabbit HA monoclonal antibody diluted 1/1000 was used for the Western Blot step.

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Xenopus laevis oocytes were injected with mRNA for HA-tagged human BORIS. Chromatin was prepared according to the Abcam X-ChIP protocol. Oocytes were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 20µl of Protein A/G sepharose beads, and 3µg of ab9110 (anti-HA, light blue) or, 3µg of ab18337 (anti-Boris, dark blue). A non-specific antibody was used as a control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).

Chromatin was prepared according to the X-ChIP protocol. Mouse embryonic stem whole cell lysate treated with disuccinimidyl glutarate (cross-linking agent). ChIP was performed using ab9110 at 1/200 dilution for 16 hours at 4°C in RIPA diluent. The bound DNA was quantitated by real-time PCR. Negative control: The parent cell line. Positive control: A cell line, which stably express an HA-tagged RARgamma protein.

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Arrb2 depends on Gβγ to induce membrane translocation of PKCα

Xenopus embryos were injected with 500 pg pkcα-gfp RNA and co-injected as indicated above the images. Animal Caps were prepared at stage 10 and immunostained as indicated. Nuclei were stained with Hoechst 33258 (blue). Images show representative results from at least two independent experiments with a minimum of six Animal Caps per experiment. Scale bars: 50 µm.

The inhibitory effect of Arrb2 MO1 (E) on PKCα-GFP membrane translocation was rescued by (F) co-injection of HA-Gβ and HA-Gγ mRNA (anti-HA (red): F′, merge: F").

HA was detected with ab9110.

(After Figure 2 of Seitz et al)

ab 9110 at a 1/200 dilution staining the mouse olineu cell line (oligodendrocyte precursor cell) by immunocytochemistry. The antibody was incubated with the cells for 30 minutes and then detected using a Cy5 conjugated goat anti-rabbit antibody.

This image is courtesy of an Abreview submitted by Katarina Trajkovic on 15 March 2006

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