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RTA Colorimetric Kit: ELISA-Spot
Item#: RTA1002
Our Price:
4 Chips For $312
25 Chips For $834

Main Features 

The three-dimensional microarray substrate captures and protects proteins in a porous membrane. Proteins printed onto the array surface enter the membrane and maintain their integrity, providing increased sensitivity and assay consistency as well as high-binding capacity. A key advantage of the z-grip system and the 3-dimensional protein binding surface is the extremely high and stable antigen-binding capacity. This binding capacity allows a distinct sensitivity advantage based on equilibrium dynamics. It is recommended that the first step in assay development be a titration of antigen concentration, starting at very high concentrations. Initial starting concentrations should be 10-1,000 higher than those used for a comparable ELISA assay. If the cost of antigenic material is a concern, it is recommended that after the initial trials, one moves quickly to a microspot format (1/2-20 nl) at the same concentration.

Standard protocol:

1. Dilute antigens in the spotting/printing buffer (provided). Suggestion: use much higher concentrations than used for plating on polystyrene; antigen concentrations can be as high as 0.10 mg/ml. Increased antigen concentrations may increase assay sensitivity. See printing section below for more details.
2. After printing, allow z-Grip chips to dry (drying at 4-8 C with 60-80% humidity can improve spot morphology; do not move chips until after the spots are dry)
3. Printed microtiter wells and slides are stable at room temperature for 3-12 months (depending on the antigen). For processing, place printed z-Grip slides individually into empty incubation dishes (Petri dishes).
4. Add 200 l z-blocker (provided) to each microtiter well or
add 10 ml Zeta-Blocker to each incubation dish and gently agitate at room temperature or 37 C for 20-60 min (depending on assay).
5. Add a user-determined amount (or titrate) of sample or control (primary antibody) solution into individual wells and incubate with gentle agitation for 20-60 minutes. at room temperature or 37 C.
6. Appropriately discard the sample or control solution.
7. Wash by performing three quick rinses with wash buffer. In some cases, this wash step can be omitted.
8. Empty incubation dishes and appropriately dispose of wash solutions.
9. Add 10 ml of 1 wash buffer to each incubation dish.
10. Add alkaline phosphatase-conjugated secondary antibody (suggested dilution range is 1:500-5,000) to each incubation dish
11. Incubate at room temperature with gentle agitation for 20 or 60 min.
12. Discard the solutions from the incubation dishes.
13. Wash by performing 2-10 quick rinses with wash buffer (provided).
14. Freshly prepare developer by adding 10 l NBT-BCIP/1 ml detection buffer (provided). Use cold.

Add 10 ml developer to each chip or 100 l to each microtiter well. Incubate chips at room temperature in the dark (e.g., in a drawer) for 12 or more minutes. (Note: For quantitative work, pre-determine development time by conducting pilot study. It is important that all chips to be compared are developed for the same time.) Developing the chips too long can result in increased background and signal saturation.
15. Discard Zeta-Developer and rinse with COLD tap water (holding under a faucet is okay). OPTIONAL: cover the chips with cold water for 2 minutes to stop further color development. Discard water.
16. Air-dry chips by positioning to allow excess water to run off. Signal will improve when dry.
17. Scan the slides and analyze data according to the Scanning and Data Analysis instructions.

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