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Synthetic peptide conjugated to KLH derived from within residues 150 - 250 of Human Myeloperoxidase.
(Peptide available as ab45976.)
Our Abpromise guarantee covers the use of ab45977 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 5 - 10 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 84 kDa (predicted molecular weight: 84 kDa).Can be blocked with Myeloperoxidase peptide (ab45976).|
|IHC-P||Use a concentration of 1 µg/ml.|
ab45977 stained in HL60 cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab45977 at 5µg/ml and ab72791 (Mouse monoclonal to alpha Tubulin - Loading Control) at 1ug/ml overnight at +4°C. The secondary antibodies used were ab150081 used at 1 ug/ml (colored green) and ab150116 (pseudo-colored red) used at 2ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab45977 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406
ab23106 staining p53 in Mouse bone marrow cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde and permeabilized in 0.1% Triton X-100 prior to blocking in 5% Goat serum for 2 hours at 25°C. The primary antibody was diluted 1/500 in PBS and incubated with the sample for 9 hours at 4°C. The secondary antibody was Alexa Fluor® 488-conjugated Goat anti-Rabbit polyclonal, diluted 1/500.
Nuclei were counterstained blue with DAPI.
Immunohistochemistical detection of Myeloperoxidase using antibody ab45977 on formaldehyde-fixed paraffin embedded Marmoset spleen sections. Antigen retrieval step: heat mediated in citric acidpH6 HIER. Permeabilization: No Blocking step: 1% BSA for 10 mins at room temperature. Primary antibody diluted at 1/600 and incubated for 2 hours in TBS/BSA/azide. Secondary antibody: anti Rabbit IgG conjugated to biotin (1/200). Submitted image shows clear scattered cytoplasmic and (possibly secreted) positivity within the red pulp. Also, the blood vessel extending from upper middle to lower left of the image, shows clear, though lighter, granular positivity within the cytoplasm of 5 cells. Taken together, this is the pattern of positivity that I would expect.
ICC/IF image of ab45977 stained HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab45977, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
The mature myeloperoxidase protein is a tetramer of two heavy chains (60 kDa) and two light chains (12 kDa). Our immunogen sequence is within the myeloperoxidase light chain. In HL60 cells, ab45977 detects bands at approximately 84-kDa, corresponding to the expected MW of full-length Myeloperoxidase protein, and at 12-kDa, corresponding to the expected MW of myeloperoxidase light chain.