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Synthetic peptide within Human Hsp27 aa 100-200. The exact sequence is proprietary.
(Peptide available as
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
This product is a recombinant rabbit monoclonal antibody.
Our Abpromise guarantee covers the use of ab109376 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/1000 - 1/10000. Predicted molecular weight: 23 kDa.Can be blocked with Hsp27 peptide (ab215449).|
|IP||1/10 - 1/100.|
|IHC-P||1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.|
|Flow Cyt||1/10 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Overlay histogram showing HAP1 wildtype (green line) and HAP1-HSPB1 knockout cells (red line) stained with ab109376. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab109376, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.
A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-HSPB1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Hsp27 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab109376 observed at 27 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109376 was shown to specifically react with Hsp27 in wild-type cells as signal was lost in Hsp27 knockout HEP1 cells. Wild-type and Hsp27 knockout samples were subjected to SDS-PAGE. Ab109376 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling Hsp27 with purified ab109376 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).
Secondary Only Control: PBS was used instead of the primary antibody as the negative control.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"