Prepare 1X Detection Antibody Cocktail as follows:
For running only 1 slide: Dilute 150 µl of 10X Detection Antibody Cocktail with 1350 µl of Array Diluent Buffer.
For running 2 slides: Dilute 300 µl of 10X Detection Antibody Cocktail with 2700 µl of Array Diluent Buffer. Keep on ice.
Insert the metal clip into the groove in the gasket and rotate the clip into the locked position. Ensure that the clip is on the same side as the orientation line on the slide.
NOTE: one of the clips has a small dot etched onto the upper rib to assist with pad designation (see slide assembly photos).
Add 100 µl Array Blocking Buffer to each well and cover with sealing tape. Incubate for 15 minutes at room temperature on an orbital shaker.
NOTE: Do not allow the pads to dry out at any time during the assay.
Dilute and combine LumiGLO® and Peroxide reagents immediately before use (to make 10 ml of a 1X solution, combine 9 ml deionized water with 0.5 ml of 20X LumiGLO® and 0.5 ml of 20X Peroxide).
Note for Kodak® Biomax® film users: This dilution of LumiGLO®/Peroxide may necessitate very short exposure times (2-3 seconds) for some targets. For more convenient exposure times (20‑30 seconds) add 20 ml of deionized water to the 10 ml LumiGLO®/Peroxide mix to make a 3 fold more diluted chemiluminescent reagent.
Immediately capture an image of the slide using a digital imaging system capable of detecting chemiluminescent signals. If desired, quantify spot intensities using commercially available array image analysis software. Alternatively, chemiluminescent film may be used. Expose film for 2 – 30 seconds using even and light pressure on the top of the development cassette (do not fasten the cassette clamps) to avoid squeezing out the LumiGLO®/ Peroxide reagent. Develop the film using an automated film developer.
NOTE: If both slides are being used, it is not recommended to expose them simultaneously in the same development cassette. In this case, leave the second slide in the wash buffer (step 12) while proceeding with steps 13 – 18 using the first slide. After the first slide is finished, proceed with steps 13 – 18 using the second slide and freshly diluted LumiGLO®/Peroxide reagent.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.
Kodak® and BioMax® are trademarks of Eastman Kodak Company.
posted November 2013