Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 10)

be inactivated with exposure to direct light. Warm the HRP color
development buffer to RT prior to addition of the color reagent
solutions. HRP color reagents A and B should be kept at their
proper storage temperature prior to mixing to maximize their
activity and prolong their shelf life. Protect the color development
solution from light.
2. Color development - Immerse the membrane in the color develop-
ment solution.* Protein concentrations greater that 100 ng will
immediately become visible as purple bands or dots. Lower
concentrations of protein will take longer, but should be visible
within 30 minutes. Avoid development periods longer than 45
minutes as bleaching or fading of color in positive reactions will
occur. If a heavy precipitate forms in the color development
solution, a fresh solution should be prepared and used immediately.
3. Washing - Stop the development by immersing the membrane in
dd water for 10 minutes with gentle agitation. Change the water at
least once during the 10 minute period to remove residual color
development solution.
4. Reading, drying and storage - Take photographs while the
membrane is wet to enhance the purple color. Acceptable
photographs can be produced using Polaroid Type 108, Polacolor 2
Land Film, at f8 and 1 second exposure. Film should be developed
for 1 minute. Dry the membrane on filter paper and store between
polyester sheets. Protect from light to minimize fading.
* Save used color development solution for testing in case results are
unsatisfactory. See Section 3.1.
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2. Color development - Immerse the membrane in the color develop-
ment solution. ** Protein concentrations greater than 100 ng will
immediately become visible as purple bands or dots. Lower
concentrations of protein will take longer, but should be visible
within 30 minutes. To maximize sensitivity, the incubation in color
development solution can be extended to 4 hours. Should a large
amount of precipitate form before color development is complete,
decant the color development solution and add additional, freshly
prepared, color development solution. The precipitate, which is
usually generated by high concentrations of alkaline phosphatase
on the membrane surface, will settle on the membrane and can
produce unusually high backgrounds.
3. Washing - Stop the development by immersing the membrane in
dd water for 10 minutes with gentle agitation. Change the water at
least once during the 10 minute period to remove residual color
development solution.
4. Reading, drying, and storage - Take photographs while the
membrane is wet to enhance the purple color. Acceptable
photographs can be produced using Polaroid Type 108, Polacolor 2
Land Film, at f8 and 1 second exposure. Film should be developed
for 1 minute. Dry the membrane on filter paper and store between
polyester sheets. Protect from light to minimize fading.
** Save used color development solution for testing in case results are
unsatisfactory. See Section 3.1.
Procedure for Horseradish Peroxidase Blots
1. Developer preparation - Add 600 µl HRP color reagent B to 100
ml of 1x HRP color development buffer at RT. Add this solution to
20 ml of HRP color reagent A immediately prior to use. This
solution cannot be successfully stored. HRP color development
solution will precipitate out of solution if kept at 0-4 °C, and will
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