GST Detection Module with CDNB Enzymatic Assay
GST-tagged proteins produced using pGEX vectors can be detected enzymatically using the GST substrate CDNB, included in the GST Detection Module. The GST-mediated reaction of CDNB with glutathione produces a conjugate that is measured by absorbance at 340 nm using either a plate reader or a UV/Vis spectrophotometer. Assay results are available in less than 10 min for crude bacterial lysates, column eluates, or purified GST-tagged protein. Figure 4.3 shows typical results from a CDNB assay. Each GST Detection Module contains reagents sufficient for 50 assays.
Fig 4.3.Typical results of a CDNB assay for GST-tagged proteins. 53 µg of total protein from an E. coli TG1/pGEX-4T-Luc lysate and 0.8 µg of total protein eluted from Glutathione Sepharose medium were assayed according to instructions included with the GST Detection Module.
CDNB is toxic. Avoid contact with eyes, skin, and clothing. In case of accidental contact, flush affected area with water. In case of ingestion, seek immediate medical attention.
pGEX-bearing cells must be lysed prior to performing a CDNB assay.
Procedure
- In a microcentrifuge tube, combine the following:
2. Cap the tube and mix the contents by inverting several times.
CDNB may cause the solution to become slightly cloudy. However, the solution should clear upon mixing.
3. Transfer 500 µl volumes of the above CDNB solution into two UV-transparent cuvettes labeled sample and blank. Add sample (5 to 50 µL) to the sample cuvette. To the blank cuvette, add 1× reaction buffer equal in volume to that of the sample in the sample cuvette
4. Cover each cuvette with wax film and invert to mix.
5. Place the blank cuvette into the spectrophotometer and blank at 340 nm. Measure the absorbance of the sample cuvette at 340 nm and simultaneously start a stopwatch or other timer.
6. Record absorbance readings at 340 nm at 1 min intervals for 5 min by first blanking the spectrophotometer with the blank cuvette and then measuring the absorbance of the sample cuvette.
7. Calculate the A340/min/mL sample as follows:
A340/min/ml values can be used as a relative comparison of GST-tagged protein content between samples of a given tagged protein.
Adapt the assay to give absolute concentrations of tagged proteins by constructing a standard curve of ∆A340/min versus amount of tagged protein. Purified sample of the tagged protein is required to construct the curve.
The assay detects active GST. Additional inactive GST-tagged protein may be present.
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