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cfps-strep-purification-thermal-shift.md

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1# Protein Expression and Thermal Shift Assay

3**URL:** https://cloud.ginkgo.bio/protocols/cfps-strep-purification-thermal-shift

4**Service terms:** https://cloud.ginkgo.bio/terms/cfps-strep-purification-thermal-shift

5**Reviewed:** 2026-09-30; prices and turnaround below are catalog estimates.

6**Status:** Ginkgo Certified

7**Price:** $159/sample

8**Turnaround:** up to 12 days

9**Throughput:** Up to 88 constructs per run (1 column reserved for controls)

Protein Expression and Thermal Shift Assay

URL: https://cloud.ginkgo.bio/protocols/cfps-strep-purification-thermal-shift Service terms: https://cloud.ginkgo.bio/terms/cfps-strep-purification-thermal-shift Reviewed: 2026-09-30; prices and turnaround below are catalog estimates. Status: Ginkgo Certified Price: $159/sample Turnaround: up to 12 days Throughput: Up to 88 constructs per run (1 column reserved for controls)

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11## Overview

13Combines cell-free protein expression, Strep-tag magnetic bead purification, and a Protein Thermal Shift Assay using SYPRO Orange into a single end-to-end workflow. Starting from a DNA template plate, the protocol expresses protein in a CFPS reaction, purifies the tagged product via Strep-Tactin magnetic beads, and characterizes thermal unfolding of the purified protein by extrinsic fluorimetry.

15SYPRO Orange fluorescence tracks exposure of hydrophobic regions during a temperature ramp. The service reports **Tonset** and up to three apparent transitions (**TM1, TM2, TM3**) where callable. Compare variants under matched conditions and review thermograms and QC flags; multiple peaks alone do not establish which structural domains unfolded. This assay supports stability ranking, not binding-affinity measurement.

Overview

Combines cell-free protein expression, Strep-tag magnetic bead purification, and a Protein Thermal Shift Assay using SYPRO Orange into a single end-to-end workflow. Starting from a DNA template plate, the protocol expresses protein in a CFPS reaction, purifies the tagged product via Strep-Tactin magnetic beads, and characterizes thermal unfolding of the purified protein by extrinsic fluorimetry.

SYPRO Orange fluorescence tracks exposure of hydrophobic regions during a temperature ramp. The service reports Tonset and up to three apparent transitions (TM1, TM2, TM3) where callable. Compare variants under matched conditions and review thermograms and QC flags; multiple peaks alone do not establish which structural domains unfolded. This assay supports stability ranking, not binding-affinity measurement.

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17## Input

19- **DNA Input:** Linear DNA sequence

20- **Tag Orientation:** N-terminal or C-terminal fusion (validated with C-terminal tags; success is protein-dependent)

21- **Format:** Up to 88 constructs per run, 1 column of wells for controls

Input

DNA Input: Linear DNA sequence
Tag Orientation: N-terminal or C-terminal fusion (validated with C-terminal tags; success is protein-dependent)
Format: Up to 88 constructs per run, 1 column of wells for controls

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23## Output

25- **Cell-Free Reaction:** 100 uL E. coli-based CFPS reaction per sample with process controls

26- **Yield Quantification:** Absolute protein yield in eluate via A280, converted to mg/mL

27- **Thermal Stability Measurement:** SYPRO Orange thermal shift in triplicate on purified eluate, reporting Tonset and up to three melting transitions (TM1, TM2, TM3) in deg C, where applicable

28- **Reporting & Data:** PDF report with yield data, thermogram plots, called TM values, reaction condition metadata, and QC status

Output

Cell-Free Reaction: 100 uL E. coli-based CFPS reaction per sample with process controls
Yield Quantification: Absolute protein yield in eluate via A280, converted to mg/mL
Thermal Stability Measurement: SYPRO Orange thermal shift in triplicate on purified eluate, reporting Tonset and up to three melting transitions (TM1, TM2, TM3) in deg C, where applicable
Reporting & Data: PDF report with yield data, thermogram plots, called TM values, reaction condition metadata, and QC status

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30## Automated Workflow