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Synthetic peptide conjugated to KLH derived from within residues 150 to the C-terminus of Human Glutathione Peroxidase 1.
(Peptide available as ab25301.)
Our Abpromise guarantee covers the use of ab22604 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||1/300 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 22 kDa (predicted molecular weight: 22 kDa).|
|ICC/IF||Use a concentration of 5 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
ab22604 staining mouse Cor-1 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with TBS/BSA/azide/0.1%Tween 20 and blocked with 1% BSA for 10 minutes at RT. Samples were incubated with primary antibody (1/300) for 2 hours. An diluted (1/1000) Alexa Fluor® 594-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: GPX1 knockout HAP1 cell lysate (20 µg)
Lane 3: THP1 cell lysate (20 µg)
Lane 4: HL60 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab22604 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab22604 was shown to recognize Glutathione Peroxidase 1 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Glutathione Peroxidase 1 knockout samples were examined. Wild-type and Glutathione Peroxidase 1 knockout samples were subjected to SDS-PAGE. ab22604 at a concentration of 1µg/ml and ab8245 (loading control to GAPDH) diluted at 1/2000 were incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
Image courtesy of Human Protein Atlas
ab22604 staining Glutathione Peroxidase 1 in Human kidney. The paraffin embedded human kidney tissue was incubated with ab22604 (1/375 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab22604 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.
Further results for this antibody can be found at www.proteinatlas.org
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab22604 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
ab22604 detects a band of approximately 22 kDa in human liver lysate. The band detected in the mouse liver lysate runs slightly higher (~ 24kDa). We believe that these bands correspond to Glutathione Peroxidase 1 as they are both blocked by the addition of the immunizing peptide (ab25301).