The 6His check kit Gold enables the rapid detection of 6His tagged fusion proteins.
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The 6His check kit Gold enables the rapid detection of 6His tagged fusion proteins.
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Overview

The kit enables the rapid detection of 6His tagged fusion proteins at each step of protein production and purification. It can either be used to ensure that the 6His motif of a fusion protein is accessible to the anti-6His antibody, or to approximate the concentration of a 6His-tagged protein.

Calibrated using the appropriate protein, the assay is very accurate and compatible with the SOPs, it can also be used to eliminate false positives and to predict the possibility of purifying the protein using a Nickel column: a non-detected Tag using our check kit will not be available for purification.

Assay principle

As shown in the graph, 6His peptide labeled with XL665 is detected by anti 6His Cryptate conjugate (anti-tag Cryptate). The 6His-tagged fusion protein (Tag fusion protein) to be ascertained competes with the 6His-peptide-XL665 conjugate (Tag-XL665) for binding to the anti-6His-Cryptate conjugate. The specific signal is inversely proportional to the concentration of 6His-tagged fusion protein.

Assay protocol

The example on the right describes the protocol using a 20 µL final assay volume. Dispense 10 µL of standard or of 6His-tagged protein of interest , with 5 µL of 6His Eu-Cryptate conjugate and 5 µL of 6His-XL665 conjugate into a well. Incubate for 2 hours at RT and read.

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

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 insertion6His Tagged proteinMW(Da)Quantification limit or IC10(nM)IC50 (nM)
N-term6His-peptide1,3004.232.8
6His BRD4(1)17,80017.6105.0
6His-SNAP20,60031.4244.8
6His-MBP-GST-Flag*
64,0003.228.0
InternalTHX-6His-1509V3*
27,0003.024.8
C-term6His tagged Nanobody15,0002.924.48
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