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Horseradish peroxidase linked anti rabbit igg

Manufactured by Cytiva

Horseradish peroxidase-linked anti-rabbit IgG is a secondary antibody conjugate used in various immunoassay techniques. It contains antibodies raised in another species that are specific to rabbit immunoglobulin G (IgG) and are covalently linked to the enzyme horseradish peroxidase.

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2 protocols using horseradish peroxidase linked anti rabbit igg

1

Protein Extraction and Western Blot Analysis

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They were performed as described in [25 (link)]. Briefly, cells were washed twice with ice-cold 1X PBS and proteins were extracted with 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40 supplemented with 50 mg/ml leupeptin, 50 mg/ml aprotinin, 0.5 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl fluoride (Sigma). Breast tumor samples were homogenized in the same lysis buffer with a Polytron homogeneizer. Protein extracts were quantified using bicinchoninic acid protein assay reagent (Pierce), resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies recognized human specific forms of GAPDH (Abcam) and phospho-STAT3 (Y705) (R&D). Secondary antibodies included horseradish peroxidase-linked anti-rabbit IgG and anti-mouse IgG (Amersham GE Healthcare). Proteins were detected with Immobilon western chemiluminescent HRP substrate (Millipore). Signals in Western blots were quantified with the software ImageJ 1.38 (NIH, Bethesda, MD, USA).
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2

Immunoblotting Analysis of Signaling Proteins

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Antibodies used were anti-phospho-AKT(Ser 473), anti-phospho-AKT(Thr308), and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (Cell Signaling Technology, Danvers, MA), anti-Akt (pan) Rabbit mAb, p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb(Cell Signaling Technology, Danvers, MA), anti-PARP rabbit mAb and Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (Cell Signaling Technology, Danvers, MA), anti-alpha tubulin monoclonal horseradish peroxidase conjugate (Cell Signaling Technology, Danvers, MA), Secondary antibodies (horseradish peroxidase-linked anti-rabbit IgG and anti-mouse IgG antibodies) were obtained from Amersham Biosciences (Uppsala, Sweden).
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