Minipreps Using GST SpinTrap™

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

GST SpinTrap™ columns are designed for rapid, small-scale purification of GST-tagged proteins using conditions of mild affinity purification. Greater than 90% purity can be achieved in a single step. The columns are suitable for purification of multiple samples in parallel, for example, expression screening experiments or optimization of purification conditions.

Each microspin column contains 50 µl of Glutathione Sepharose® 4B, enough to purify up to 500 µg of recombinant GST (rGST). The capacity will vary with the nature of the GST-tagged protein and the binding conditions used. Refer to Appendix 2 (Characteristics of Glutathione Sepharose® Products) for the main characteristics of GST SpinTrap.

GST SpinTrap

Fig. 5.5. GST SpinTrap™ is a single-use column for rapid, small-scale purification of GST-tagged proteins.

 

Sample preparation

For small-scale cultures, freeze/thaw or chemical lysis with commercial kits are recommended for cell lysis. GE can provide lysis kits for different expression systems: Mammalian Protein Extraction Buffer for mammalian expression systems and Yeast Protein Extraction Buffer Kit for yeast expression systems. For bacteria, several chemical lysis kits are available on the market.

Adjust the sample to the composition and pH of the binding buffer by additions from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange.

Pass the sample through a 0.22 µm or a 0.45 µm filter, and/or centrifuge it immediately before sample application. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.

Note: Cell culture lysates may also be directly applied to the column without prior clarification.


Buffer preparation

Recommended buffers can easily be prepared from GST Buffer Kit.

Binding buffer: 10 mM PBS, pH 7.4 (10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4 pH 7.4)
Elution buffer: 50 mM Tris-HCl, 10 to 20 mM reduced glutathione, pH 8.0

1 to 20 mM DTT may be included in the binding and elution buffers to reduce the risk of oxidation of free -SH groups on GST, which may cause aggregation of the tagged target protein, resulting in lower yield of GST-tagged protein.


Purification

The capacity of each SpinTrap™ column is 500 µg of GST-tagged protein. A maximum of 600 µl of culture lysate or buffer can be applied to a SpinTrap™ column. This represents roughly a volume of lysate produced from a 12 ml culture. The following procedure is designed for lysates prepared from 2 to 12 ml of culture.

Perform purifications on GST SpinTrap™ using a standard microcentrifuge. Place the column in a 2 ml microcentrifuge tube to collect the liquid during centrifugation. Use a new 2 ml tube for every step (steps 1 to 5).

  1. Invert and shake the column repeatedly to resuspend the medium. Loosen the top cap one-quarter of a turn and twist/break off the bottom closure. Place the column in a 2 ml microcentrifuge tube and centrifuge for 30 s at 100 × g (approx. 1500 rpm in an Eppendorf 5415R, 24-position fixed-angle rotor) to remove the storage liquid.
  2. Remove and discard the top cap. Equilibrate the column by adding 600 µl of binding buffer. Centrifuge for 30 s at 100 × g.
  3. Add the sample (see Sample preparation). The maximum sample volume is 600 µl per column. Centrifuge for 30 s at 100 × g. Mix gently at room temperature for 5 to 10 min to ensure optimal binding of GST-tagged proteins to the Glutathione Sepharose® 4B medium.

It is possible make several sample applications as long as the binding capacity of the column is not exceeded.

  1. Wash with 600 µl of binding buffer. Centrifuge for 30 s at 100 × g. Repeat the wash step once.
  2. Elute the target protein twice with 200 µl of elution buffer. Centrifuge for 30 s at 100 × g, and collect the purified sample. The first 200 µl will contain the majority of the target protein.

Yields of GST-tagged protein may be increased by repeating the elution step two or three times and pooling all eluates.

 

Materials

     
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