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Full length native protein (purified) corresponding to Human Hsp27. (Partially purified human HSP27)
Our Abpromise guarantee covers the use of ab2790 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|ICC/IF||Use a concentration of 0.5 - 1 µg/ml.|
|IHC-P||Use a concentration of 0.5 - 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use citrate buffer.
Immunocytochemistry/ Immunofluorescence analysis of non small cell lung carcinoma NCI-H1299 cells labeling Hsp27 with ab2790 at 1/300 dilution. The cells were fixed with formaldehyde and permeabilised with 0.2% TritonX-100. The cells were blocked with 1.5% serum for 10 minutes at 25°C, followed by incubation with Anti-Hsp27 antibody [G3.1] (ab2790) in 1x HBSS + 0.02% TritonX-100 + 1.5% FBS for 3 hours at 25°C. A polyclonal goat anti-mouse IgG Alexa Fluor® 488 was used as the secondary antibody at 1/1000 dilution. DAPI nuclear stainining.
Immunohistochemistry of paraffin embedded human breast carcinoma with ab2790 labeling Hsp27 at 0.5μg/ml.
Immunohistochemistry of paraffin embedded human prostate carcinoma with ab2790 labeling Hsp27 at 0.5μg/ml.
Cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/200 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594 (ab150080), was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in
Overlay histogram showing HeLa cells (ab150035) stained with ab2790 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum (ab7481) / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2790, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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.
ab2790 staining Hsp27 in A549 lung cancer cells by Flow Cytometry. Cells were harvested in trypsin, fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100. The sample was incubated with the primary antibody (1/50 in 1x HBSS + 0.02% Triton X-100 + 1.5% FBS) for 3 hours at 25°C. An Alexa Fluor® 488-conjugated goat anti-mouse IgG polyclonal (1/2000) was used as the secondary antibody. Gating Strategy: No gating.
Flow Cytometry analysis of mouse lewis lung carcinoma cells labeling Hsp27 with ab2790. Cells were harvested in trypsin, then fixed with praformaldehyde and permeabilized with 0.2% Triton X-100. Followed by incubation with Anti-Hsp27 antibody [G3.1] (ab2790) at 1/200 dilution in 1x HBSS + 0.02% Triton X-100 + 1.5% FBS for 3 hours at 25°C. A polyclonal goat anti-mouse IgG Alexa Fluor® 488 secondary antibody was used at 1/2000.