Way to Go, Indigo! The Basic Assay
Adapted from Diane Sweeney, “Way to Go, Indigo!” Exploring Life Lab Manual © 2002
Special thanks to Mr. Art Roberson, Technical Sales Manager at Genencor International,
for donating IndiAge Super GX Plus enzyme and providing technical assistance!
Materials
Equipment per team Reagents & consumables
Day 1. scissors Day 1. three 3 in x 3 in dark denim fabric swatches
Sharpie™ marker IndiAge solution (2% enzyme in pH 5 buffer)
two 250-mL beakers pH 5 buffer
graduated cylinders Ziploc™ bags
goggles & aprons label tape
envelope
Day 2. plastic wash basins Day 2. paper towels
magnifying glass or stereomicroscope sponges
400 mL plastic beakers disposable gloves
goggles & aprons
3 large water baths set to 55°C
Day 1.
1. Obtain a 3 in x 9 in piece of dark denim and cut it into thirds, producing three 3 in x 3 in swatches.
2. To distinguish the experimental denim swatch (the one you will treat with IndiAge), cut off one of its corners. With a marker, write “C2” (for control 2) on the back of a second denim swatch and set it aside until step 11. Leave the third swatch, control 1, unmarked.
3. With masking tape and a permanent ink marker, label one beaker or large cup, “Buffer with IndiAge.” Label a second beaker or cup, “Buffer Only”
4. In each beaker, stand up a small Ziploc™ bag. Pour 200 mL of Buffer with IndiAge solution into the bag in the “Buffer with IndiAge” beaker, and 200 mL of pH 5 buffer only into the “Buffer” bag.
5. Place the denim swatch with the cut corner in the bag in the “Buffer with IndiAge” beaker. Place the unmarked (control 1) swatch in the beaker labeled “Buffer.” Push air out and zip both bags closed.
6. Allow the two denim swatches to incubate at room temperature for 3-4 days.
Day 2.
7. Remove both swatches from their beakers. Keeping them SEPARATE, rinse the swatches WELL under HOT running water. NOTE: The baggies’ liquids must be neutralized with baking soda before disposal.
In denim processing facilities, jeans are placed in industrial washers to break the hydrolyzed cellulose fibers and release
indigo molecules. We’ll hand wash our swatches with a sponge to simulate this process!
8. Put on plastic gloves so your hands don’t turn blue. J
9. Put the denim swatches in separate containers of about 400 mL hot water (don’t burn your hands!). Scrub the pieces with sponges in the water, and lift them out and squeeze repeatedly. Continue scrubbing the swatches for 10 full minutes. Change the water whenever it gets blue. Try to give each swatch the same all-over scrubbing and squeezing treatment.
10. When no more blue dye is released (or after 10 minutes), wring out the swatches one last time and lay them flat on a paper towel to dry overnight.
11. Compare all three denim swatches when dry. If time permits, use a magnifying glass or stereoscope to look for differences in the fibers. What macroscopic or microscopic effect(s) did IndiAge have?