Question (41630) | Human EGF ELISA Kit (ab100504)

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Question

Dear tech support
We recently purcahsed ELISA kits for human EGF ab100504. While doing an
assay on one of the kits that we ran into a hiccup despite following the
protocol to the point. The standard curve did not work well with black
precipitates in the wells. COuld you advice what may be the problem?the
standards were prepared as directed in the recommended protocol ... 0.82
pg/ml to 200 pg/ml.
Many thanks

Answer

Thank you for contacting us and reporting the problems you have been encountering with the EGF Human ELISA Kit (ab100504). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

If you don't mind, could I ask a few questions in order to understand the situation a bit better.

1. How had the standard been handled prior to use? How long before carrying out this experiment was it reconstituted and how was it stored between reconstitution and testing?

2. Were the reagents allowed to cone to room temperature prior to use?

3. After which step did the precipitate begin to form? How quickly did it form?

4. Was there less precipitate with less standard present (i.e. was there no precipitate in the 0 standard well)?

5. Did you observed any precipitate in the HRP-streptavadin concentrate? Was this thoroughly mixed prior to use?

6. At what temperature was the HRP-streptavadin incubated with the plate? Was this performed in the light?

7. Was the TMB substrate reagent incubated in the dark?

I am sorry for all the questions but hopefully this information will help me to understand why this precipitate is forming. If you could also provide me with the order number and the lot number of the kit that would be very useful.

I look forward to receiving your reply.

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