The production of fusion proteins requires the confirmation of their structural integrity and the accessibility of the associated tag following isolation. The GST and 6HIS check kits are simple tools for assessing tagged proteins during production processes and drug discovery.
The GST and 6HIS check kits are competitive immunoassays that use Cryptate-labeled anti-tag antibodies and XL665-labeled tags. The check kit assays can be run in a single microplate, and require only a two hour incubation. Because of the ratiometric measurement of HTRF, tagged proteins can be accurately detected in a variety of buffers and media.
A fast, simple HTRF protocol makes it possible to measure GST or 6-HIS tagged proteins at each step of protein production and purification. These kits can be calibrated using the appropriate protein, making the assay very accurate and compatible with the SOPs used in industrial processes.
6HIS and GST check kits are useful after HTS to eliminate false positives. Compounds that interrupt the binding of tag-detection reagents (e.g. HTRF MAb Anti-6HIS-Cryptate) will show strong signal inhibition in the check kit. These compounds can be removed from further characterizations, saving time and resources.
The 6HIS check kit is a useful tool to determine whether the tag is accessible to antibodies and to predict the possibility of purifying the protein using a Nickel column. If a 6HIS fusion protein is not detected using our check kit, then the tag is inaccessible and not available for purification or other binding purposes.
| Detection limit: | 1 ng/mL (average) |
|---|---|
| S/B | 15 in 96- or 384-well formats |
A standard curve using native GST was performed following the protocols detailed in the product inserts. The detection limit of this assay was 0.26 ng/ml
The GST check kit enables the verification of tag accessibility on GST-tagged fusion proteins. The figure shows the dose response curves of different GST fusion proteins assessed using this kit. All six GST containing proteins were detected using our competitive immunoassay.
GST check assay was performed using the standard protocol and a series of six different GST proteins.
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