Antibody Microarray: A Technique to Study the Protein Expression of miRNA Treated Lung Cancer Cells
Transcript
– Start by adding a biotin solution and a labeling buffer to the protein sample. Incubate to facilitate attachment of biotin to the proteins. Add a stop reagent and vortex. Next, place an antibody microarray slide in a dish containing a blocking reagent to prevent non-specific binding of antibodies. Wash the slide with double distilled water to remove the blocking solution. Place the microarray slide in a chamber. Mix the biotinylated protein sample with a coupling solution and add onto the slide to facilitate the binding of the target proteins to their corresponding antibodies. Transfer the slide to a dish containing a washing buffer and replace the buffer with double distilled water. Next, place the slide in a detection buffer containing fluorescently labeled streptavidin to promote its binding to the biotinylated proteins.
Incubate the dish in the dark to maintain the fluorescence intensity. Remove the slide and place it in a microarray scanner. The scanner looks for fluorescent signals from the streptavidin bound to the target protein. Based on the fluorescence intensity, the software analyzes the extent of protein expression. In the following protocol, we perform an antibody microarray for analyzing alterations in gene expression associated with the cell cycle.
– This experiment is to identify all the cell cycle pathway proteins' expression change by the microRNA treatment. Collect protein samples according to the procedure described in the transfection section and quantify with BCA assay. Remove the slides from minus 4 degrees Celsius one hour before experiment to bring to room temperature and to remove moisture. Take 70 microgram of protein samples and prepare sample slides according to the manufacturer's instructions, which is also described step by step in the text section.
Here to note, proper washing of slides with ultrapure deionized water is another important step as this will help to reduce the background of the slides. Additionally, it is very critical in this experiment not to dry up the sample slides until the final step. Finally, scan the slide using microarray machine and analyze the data.
