Product Protocol: BMP2 antibody used to evaluate vitreous modulation of gene expression in low-passage human retinal pigment epithelial cells (OASA07470)

Specificity: BMP-2
Product Page: BMP2 antibody (OASA07470)
Antibody Pair Available: OASA07470 Capture OASA07472 Detection
Experiment Type: Immunohistochemistry, Immunoblot Analysis

1. Immunohistochemistry:

RPE cells were grown for 48 hours in control medium or medium containing 25% vitreous. To inhibit secretion of extracellular proteins, Brefeldin A (Sigma-Aldrich, St. Louis, MO) was then added to the medium to a final concentration of 5 ng/mL, and the incubation was continued for a further 6 hours. The cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with rabbit anti-human tissue plasminogen activator antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or with rabbit anti-human BMP-2 antibody (Aviva Systems Biology, San Diego, CA) followed by Cy5-conjugated anti-rabbit IgG (Invitrogen). The cells were double stained with Texas Red phalloidin (Invitrogen) to detect actin microfilaments and were observed by confocal microscopy (Meta500; Carl Zeiss Meditec, Inc., Dublin, CA).

2. Immunoblot Analysis:

The cells were extracted with ice-cold extraction buffer (1% NP40; Sigma-Aldrich) in 50 mM Tris-HCl (pH 8.0) containing 340 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM NaF and protease cocktail inhibitor (20 μL/mL; Pierce Biotechnology Inc., Rockford, IL). The extract was centrifuged and dissolved for SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories) and protein-binding sites were blocked overnight with 5% nonfat dried milk in blocking buffer (20 mM Tris-HCl [pH 7.6], 0.8 M NaCl, and 0.1% Tween-20). Membranes were incubated overnight at 4°C with the appropriate primary antibody diluted in blocking buffer. The primary antibodies used were rabbit anti-human TFPI2 (kindly given by Walter Kisiel, Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM; diluted 1:1000), or rabbit anti-human BMP-2 antibody (diluted 1:100; Aviva Systems Biology). After the membrane was washed, it was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare). Chemiluminescence was used for detection (West Pico Supersignal; Pierce Biotechnology, Inc.).

1. To determine whether vitreous modulates the levels of BMP-2 protein, RPE cells were grown in the presence or absence of vitreous for 48 hours and then treated with 5 ng Brefeldin A/mL for 6 hours to prevent secretion of BMP-2. Cells were stained using anti-human BMP-2 antibody to detect intracellular BMP-2 and were also stained for F-actin so that the proportion of cells expressing BMP-2 could be determined. Control cells showed little cytoplasmic fluorescence for BMP-2. However, vitreous-treatment caused increased levels of BMP-2 protein in virtually all cells. 2. Immunoblot analysis indicated that treatment of the cells for 42 hours with vitreous followed by 6 hours with Brefeldin A resulted in the accumulation of a protein with an apparent molecular weight of ∼42 kDa that bound anti-human BMP-2 antibodies. This size of protein was consistent with the molecular weight of pro-BMP-2.

1: Ganti R, Hunt RC, Parapuram SK, Hunt DM. Vitreous modulation of gene expression in low-passage human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2007 Apr;48(4):1853-63. PubMed PMID: 17389521.

This entry was posted in Antibody Pairs and tagged , , , , . Bookmark the permalink.

Leave a Reply