Detection of GST-tagged Proteins

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

Several methods are available for detection of GST-tagged proteins, with method selection largely depending on the experimental situation. For example, SDS-PAGE analysis, although frequently used for monitoring results during expression and purification, may not be the method of choice for routine monitoring of samples from high-throughput screening. Functional assays based on the properties of the protein of interest (and not the GST tag) are useful but must be developed for each target protein. These assays are not covered in this handbook.

Much of the information presented below can also be applied to detection of histidine-tagged proteins, although the specific reagents will change.

GST Detection Module with CDNB enzymatic assay

GST-tagged proteins produced using pGEX vectors can be detected enzymatically using the GST substrate 1-chloro-2,4 dinitrobenzene (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 sonicates, column eluates, or purified GST-tagged protein. Figure 5.16 shows typical results from a CDNB assay. Each GST Detection Module contains reagents sufficient for 50 assays.

Typical results of a CDNB assay for GST-tagged proteins.

Fig 5.16. Typical results of a CDNB assay for GST-tagged proteins. 53 µg of total protein from an E. coli TG1/pGEX-4T-Luc sonicate and 0.8 µg of total protein eluted from Glutathione Sepharose were assayed according to instructions included with the GST Detection Module.


Components of GST Detection Module used with the CDNB enzymatic assay

  • 10× reaction buffer: 1 M KH2PO4 buffer, pH 6.5
  • CDNB: 100 mM 1-chloro-2,4-dinitrobenzene (CDNB) in ethanol
  • Reduced glutathione powder: Prepare a 100 mM solution by dissolving the glutathione in sterile distilled water. Aliquot into microcentrifuge tubes. Store at -20°C. Avoid more than five freeze/thaw cycles.

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.

 

Steps

  1. In a microcentrifuge tube, combine the following:
Distilled water
10× reaction buffer
CDNB
Glutathione solution
880 µl
100 µl
10 µl
10 µl
Total volume
1000 µl
  1. 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.

  1. 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.
  2. Cover each cuvette with wax film and invert to mix.
  3. 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.
  4. 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.

For analyses using 96-well plates, add samples to the cells first and add reagents second. Mix the contents of the wells using the pipette. Start measuring the absorbance in the plate reader.

Calculate the A340/min/ml sample as follows:

∆A340/min/ml = A340(t2) – A340(t1)
(t2 – t1)(ml sample added)
  Where: A340 (t2) = absorbance at 340 nm at time t2 in min
A340 (t1) = absorbance at 340 nm at time t1 in min

∆A340/min/ml values can be used as a relative comparison of GST-tagged protein content between samples of a given tagged protein.

Western blot

Expression and purification of GST-tagged proteins can be monitored by Western blot analysis, Amersham ECL is a chemiluminescent detection reagent providing adequate sensitivity for most recombinant expression applications. For higher sensitivity, use Amersham ECL Prime or Amersham ECL Select (chemiluminescence), alternatively, Amersham ECL Plex (fluorescence). The combination of Western blot detection and total protein staining of the SDS-PAGE (using Coomassie, or silver stain) gives a powerful control of purification results. See Western Blotting: Principles and Methods, 28999897.

Materials

     
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