Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)
Figure 2
ELISA of TIMP-2, E-cadherin, RAR and CRABP: MCF-7 (300,000 cells/1.5 mL) cells were grown in serum free culture medium (SFCM) in absence (C) and in presence of 30 M ATRA (E) for 24 hours. The culture supernatants were collected and the wells of an ELISA plate was coated with 50 L SFCM (for TIMP-2) and 50 g of protein (for E-cadherin, RAR & CRABP) from both control and ATRA-treated cells and kept at 4°C for overnight. The next day contents of the wells were discarded and the wells were washed with blocking buffer. Then 50 L of blocking buffer was added to each well and kept at 37°C for 1 hour. The blocking buffer was discarded and the wells were washed with washing buffer. The wells were then reacted with anti-TIMP-2 (Figure 2), anti-Ecadherin (Figure 5(c)), anti-RAR (Figure 8(a)), anti CRABP (Figure 8(b)) antibody, respectively, and kept in 37°C for 1 hour. The wells were washed with washing buffer and reacted with respective HRP-coupled secondary antibody and kept in 37°C for 1 hour. The wells were washed with washing buffer and then TMB substrate was added to each well for color development. The color reaction was stopped with 1 M solution and OD of each well was measured at 450 nm. The OD indicated the expression level of TIMP-2 ( ()) in control and treated MCF-7 cells.
