Thursday, November 1, 2007

Multi-analyte Microarrays and ELISA

Standard ArrayIt Multi-analyte Protein Microarray Protocol

1. Print antigens at a final concentration of 0.3mg per ml as a final concentration in protein printing buffer onto SuperEpoxy 2 surface chemistry using a NanoPrint microarrayer. For colorimetric detection use SuperNitro. Consider immunoglobulin controls as a dilution series for quantitation "dose response" curves. After microarray manufacturing, let microarrays sit overnight on the deck microarrayer to dry. Stored printed microarrays clean, dry and at room temperature. Protein printing buffer will stabilize the printed proteins for long periods of time.

2. Prior to using the microarrays they must be blocked. Incubate microarray(s) in 1X blocking buffer from 1 hour to overnight. Blocking for less than 1 hour can compromise results. After blocking wash microarray 3 times 5 minutes each in PBS or PBST.

3. React primary antibody to the microarrays(s). Use diluted serum or other test sample diluted into 1X BlockIt Buffer, typical dilutions are 1 to 200. Because kinetics of microarrays are fast, incubations can be as short as 15 minutes or as long as overnight. Agitation and increased temperature can be used to speed up kinetics. Temperature can varied depending on the application from 4 C to 37 C.

4. Wash off reaction 3 time 5 minute each in 1X PBS or PBST.

5. React secondary antibody to the microarray. Secondary antibodies can be labeled with fluorescent markers for detection in microarray scanners or conjugated to AP and subsequently developed using an alkaline phosphatase development kit. Protect fluorescent reactions from light to avoid photo bleaching. Incubate 1 minute to 1 hour.

6. Wash microarray(s) in 1X PBS or PBST 3 times for 5 minutes. Rinse for 15 seconds in ddH20 and dry. A microarray centrifuge can be used to dry slide based microarrays. If using fluorescent detection, scan immediately.