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Thanks for your help and time spent on my order. I am very impressed by your quick response and work efficiency. Yes, the information to my questions will still be appreciated so I can keep updated with your product. Even this product may not fit my need, Abcam will always be my first choice for purchase.
Asked on Aug 16 2012
I understand your confusion. Some of the language of the protocol is a little difficult to understand.
The ELISA kti contains two antibodies: one that is coated onto the wells which is anti-His tag. The detector antibody in the kit is rabbit anti-GST, and this is in turn detected by an anti-rabbit IgG antibody conjugated to HRP. Addition of HRP substrate produces the colored reaction.
I think this is the part of the ELISA description that is confusing:
"Bound proteins can be detected using rabbit antibody specific to the 6XHistagged fusion protein of choice (not included)."
This is confusing, since the kit obviously will not include an antibody against a unique protein. The note is meant to suggest to users that they could use the anti-IgG secondary antibody that is included with the kit, conjugated to HRP, to detect their own rabbit antibody raised against their protein of interest, if they had such an antibody.
This is repeated in the next part of the introduction:
"Standards and samples are pipetted into the wells and the 6XHis-tag present in the sample is bound to the wells by the immobilized antibody. The wells are washed and a rabbit analyte specific detector antibody is added. After washing away unbound detector antibody, an HRP-conjugated antibody specific for the rabbit antibody is pipetted to the wells."
An analyte-specific detector antibody is not in the kit. What the kit does contain is stated in the earlier sentence:
"However, this kit does include a detector antibody and standard protein for use with dual GST 6XHis-tagged proteins." The detector antibody is anti-GST.
I hope this helps. Please let me know if you have more questions.
Answered on Aug 17 2012