I am trying to assess binding activity of a His-tagged recombinant to ganglioside GT1b via a sandwich assay (ELISA). I have performed the experiment numerous times with 2 different gangliosides and 3 different positive control proteins as well as performing with and without GT1b bound to the plate, and EVERY time my test and negative control His-tagged proteins bind to the plate regardless of whether GT1b is present or not, and my positive control proteins show baseline readings. I'm starting to go a bit crazy. I don't know what I'm doing wrong. I am following a published protocol. Does anyone have any ideas?
Assay:
GT1b in MetOH coated overnight (or just MetOH), (have also tried GT1b in PBS/BSA or PBS/skim)
Block (have tried BSA and skim milk) 1 h @ 37C
Proteins 2 h @ 37C
Primary antibody 1 h @ 37C
Secondary antibody 1 h @ 37C
Develop (HRP)
Any help would greatly appreciated, as this is one of the last experiments I need to do to finish my PhD.
Natalie
Hi Natalie
Are you using His-tagged protein...You can also cleave the His-Tag and try your assay once more...
Praveen - what am I checking for by silver staining? I have already checked them by coomassie and Western blot.
Ajay - Yes, but there is still the issue of the positive control proteins not binding.
We have recently been facing the same problem. The assay plates appeared to be the major issue. Use of vinyl assay plates from Corning Inc. (catalog no. 2595) has been successful in our hands. See Willjes et al., 2013; Biochemistry 52, 3930−3938.
Thank you for the information! It is very much appreciated. I will definitely try those plates.
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