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Split-GFP quantification is how we measure expression for your designs. We append a split-GFP11 fragment to every construct, add the complementary GFP1-10 fragment as a reagent after expression, and read the resulting fluorescence against a standard curve. The concentration reported in your results comes directly from that fluorescence.

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What we append to your design

Before expression, we add a two-part tag to your submitted sequence, joined by flexible GGGS linkers. Default placement is the C-terminus.
  1. split-GFP11, for expression quantification.
  2. Twin-Strep-tag, for purification and immobilization on the sensor.
The split-GFP11 fragment sits next to the affinity tag:
See Construct and tag design for the full appended sequence and tag placement.

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GFP11 versus GFP1-10

These are the two halves of the same fluorescent protein, but only one of them is part of your construct.
GFP1-10 is a reagent, not part of your construct. It never appears in the sequence you submit or in the protein we express. Only the GFP11 fragment is on your design.
When GFP11 on the expressed protein meets GFP1-10 in solution, the two fragments reconstitute into functional, fluorescent GFP. Neither fragment fluoresces alone, so fluorescence is proportional to the amount of GFP11-tagged protein present.

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How the measurement works

1

Express

Your design is expressed with the split-GFP11 and Twin-Strep-tag construct attached.
2

Add GFP1-10

We add the GFP1-10 detection reagent in excess to the expressed material.
3

Read fluorescence

Reconstituted GFP fluorescence is read in relative fluorescence units (RFU).
4

Convert to concentration

RFU is converted to concentration using a standard curve built from known standards.
The readout reflects total soluble expression, measured after expression.
split-GFP standard curve: measured fluorescence against GFP11-tagged protein concentration, with a linear fit of y = 130.1x + 261 and R² = 0.994 over 2.5 to 25 µM

split-GFP standard curve measured on our platform. Reconstituted fluorescence scales linearly with the amount of expressed (GFP11-tagged) protein over 2.5 to 25 µM, which is what we use to quantify expression. The blank and the 30 µM point are plotted but sit outside the fitted range.

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Fluorescence is the direct measurement

There are no intermediate absorbance or A280 readings behind the reported concentration. Fluorescence is read, the standard curve converts it, and that value is what appears in your results. We also do not run separate SDS-PAGE, SEC or A280 characterization on designs. Once expression is validated, designs go directly into the binding assay.

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Which formats carry the tag

The split-GFP tag is added to all proteins except Fabs and IgGs. Oligomeric proteins are more sensitive to tag addition, which can affect their assembly, so we leave those formats untagged. An alternative expression quantification approach for Fabs and IgGs is in development.

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FAQ

Directly from fluorescence. The GFP1-10 reagent is added after expression, reconstituted GFP fluorescence is read in RFU, and a standard curve of known standards converts that RFU value to concentration. There are no intermediate measurements between the fluorescence reading and the reported concentration: no absorbance step, no A280.
No. We add the split-GFP tag to all proteins except Fabs and IgGs. Oligomeric proteins are more sensitive to tag addition, which can affect their assembly, so those formats are expressed without it. An alternative approach for Fabs and IgGs is in development.
Yes. We do not cleave the tag between expression and binding, so the reported binding data is measured on the tagged protein.