Luminex xMAP Antibody Coupling Kit - Manual (Page 22)

Page 18 of 20
Low/No
signals
The reagents may not
have been stored
correctly
Store all reagents as recommended.
EDC may not have
been stored correctly
or rehydrated long
before use
EDC must remain in the pouch it is
shipped in until ready to use.
EDC must be used immediately in the
coupling procedure after rehydration.
The stock protein
concentration is
possibly low
Make sure the protein concentration is
correct and the recommended amount
is coupled to the microspheres.
The stock protein
suspension possibly
contains foreign
proteins, azide,
glycine, Tris or some
primary amines
Dialyze the protein of interest to
remove the competing substances.
The reaction volumes
and/or mixing method
may be incorrect
The final reaction volume for the 2 hr
incubation is critical for successful
coupling. Refer to the protocol. End-to-
end mixing during the 2 hr incubation
is very important. Please use an
optical rotator.
The anti-species
detection antibody
may not be correct
Make sure the anti-species detection
antibody is correct, for example if you
are using a mouse monoclonal to
couple to the microspheres, use an
anti-mouse detection antibody.
Make sure the anti-species detection
antibody has the appropriate label on
it, either biotin or phycoerythrin.
Problem Possible Cause(s) Recommendations