Z-138 cells were treated with 5uM of GSK-591 and Ro-0812 for 24 h were collected (20 × 106 cells were used per IP reaction) and washed twice with ice-cold PBS. Cells were lysed in ice-cold IP lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl and 0.5% NP40) for 30 min on ice and frozen at −80 °C immediately to aid the lysis. On the day of IP, the lysate was centrifuged to precipitate the debris. Supernatant was collected and incubated with 5 µg of anti-MSI2 antibody (Abcam) or Rabbit IgG (Millipore) overnight at 4 °C. RNA–MSI2 endogenous antibody complexes were pulled down using Dynabeads Protein A/G (Millipore) and washed five times in 100% IP lysis buffer, 70% IP lysis buffer and 30% PBS, 50% IP lysis buffer and 50% PBS, 30% IP lysis buffer and 70% PBS and 100% PBS. RNA was extracted using the phenol–chroloroform method and quantified by qRT–PCR.
Anti msi2 antibody
Manufactured by Abcam
Sourced in United Kingdom
The Anti-Msi2 antibody is a laboratory tool used to detect and study the expression of the Msi2 protein, which is involved in the regulation of stem cell maintenance and differentiation. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and quantify the Msi2 protein in biological samples.
Automatically generated - may contain errors
3 protocols using anti msi2 antibody
1
Immunoprecipitation of MSI2-RNA Complexes
2
Msi2 Protein-RNA Interaction Profiling
3
Quantitative Immunohistochemical Analysis of Msi2
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Immunohistochemistry (IHC) analysis was performed as previously described, 15 using anti-Msi2 antibody (Abcam, Cambridge, cat. no. ab76148). The degree of immunostaining was evaluated by two independent observers who were blind to the clinical data of the patients. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. Intensity of stained cells was graded semi-quantitatively into four levels: 0 (no staining); 1 (weak staining = light yellow); 2 (moderate staining = yellow brown) and 3 (strong staining = brown); and the proportion was scored as: 0, negative; 1, 10% or less; 2, 11-50%; 3, 51-80%; or 4, 80% or more positive cells. Intensity and fraction of positive cell scores were multiplied for each marker and thus the scoring system was defined as low expression for scores of 0-3, and as high expression for scores of 4-12.
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