6His Gold, GST, FLAG tag check kit

For detecting and measuring all GST, 6His and FLAG tagged proteins.

The production of fusion proteins requires the confirmation of their structural integrity and the accessibility of the associated tag following isolation. The GST and FLAG check kits together with 6His check kit Gold are simple tools for assessing tagged proteins during production processes and drug discovery.

Discover the new Anti-6His Check Kit Gold, to always go further in sensitivity and assay window.

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Assay Principle

These check kits are all competitive immunoassays that use Cryptate-labeled anti-tag antibodies and XL665-labeled tags. The assay can be run in a single microplate, and requires 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.

Among these, discover the new Anti-6His Check Kit Gold, to always go further in sensitivity and assay window.

Product Applications

Protein Quantification

A fast, simple HTRF protocol makes it possible to measure GST, 6His and FLAG 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.

Drug Discovery

6His Gold, GST and FLAG 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.

Tag Accessibility

The 6His check kit Gold 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.

Product Performance

1. 6His Check Kit Gold performance on various 6His-tagged proteins

The data above show the sensitivity of the 6His check kit Gold for detecting different 6His Tagged proteins. The data were generated using a PheraStar Plus reader, T1H at RT. The 6His check kit Gold enables the detection of 6His tagged proteins, whatever the molecular weight and the 6His tag insertion (N-terminal, C-terminal and internal 6His tags). The detection of the 6His tag varies for different fusion proteins. For quantitative determination of 6His tagged protein in a sample, we recommended using an internal calibrator (6His fusion protein close to the 6His tagged protein of interest).

The 6His-MBP-GST-Flag and the THX-6His-1509V3 proteins were kindly provided by Hybrigenics.

Tag insertion 6His Tagged protein MW(Da) Quantification limit or IC10(nM) IC50 (nM)
N-term 6His-peptide 1,300 4.2 32.8
6His BRD4(1) 17,800 17.6 105.0
6His-SNAP 20,600 31.4 244.8
6His-MBP-GST-Flag 64,000 3.2 28.0
Internal THX-6His-1509V3 27,000 3.0 24.8
C-term 6His tagged Nanobody 15,000 2.9 24.48
6His Tagged protein MW(Da) Quantification limit or IC10(nM) IC50 (nM)
N-term 6His-peptide 1,300 4.2 32.8
N-term 6His BRD4(1) 17,800 17.6 105.0
N-term 6His-SNAP 20,600 31.4 244.8
N-term 6His-MBP-GST-Flag 64,000 3.2 28.0
Internal THX-6His-1509V3 27,000 3.0 24.8
C-term 6His tagged Nanobody 15,000 2.9 24.48

2. GST Check Kit performance on various GST-tagged proteins

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.

3. Native GST standard curve using GST Check Kit

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.

4. FLAG Check Kit Assay Performance

The FLAG Check Kit enables the rapid detection of FLAG-tagged fusion proteins. It can either be used to ensure that the FLAG motif of a fusion protein is accessible to the anti-FLAG antibody, or to approximate the concentration of a FLAG-tagged protein.

FLAG tagged protein - 2h incubation at RT

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
GST check kit - 1,000 tests62GSTPEB
FLAG check kit - 500 tests63ADK036PEG
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FLAG check kit - 10,000 tests63ADK036PEH
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6His Check kit Gold - 1,000 tests64HISPEB
6His Check kit Gold - 10,000 tests64HISPEH

Companion products

DescriptionCat. noProduct insertMSDS
MAb Anti-6HIS-Eu cryptate Gold - 5,000 tests61HI2KLA
MAb Anti-6HIS-Eu cryptate Gold - 20,000 tests61HI2KLB
MAb Anti-6HIS Tb cryptate Gold - 5,000 tests61HI2TLA
MAb Anti-6HIS Tb cryptate Gold - 20,000 tests61HI2TLB
FLAG check kit - Standard63ADK036CDA
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