Affinity chromatography is typically done with agarose beads with particle sizes of 40-150 µm. This material is very well suited for gravity flow and FPLC applications. Cube Biotech offers PureCube and PureCube 100 agaroses with a large variety of modifications that cover most applications. However, for specialized experiments, agaroses with exceptionally large bead sizes (300-500 µm in diameter) can be useful. For example, large beads settle fast by gravity and are therefore useful for automated purification procedures. Please note that due to the significantly different surface:volume ratio, binding capacity is usually lower than for standard sized beads. The GST tag is a 26 kDa protein that binds to glutathione-coupled agarose resins. This tag is considerably larger than the His tag and is often chosen to promote recombinant protein folding.
PureCube Glutahione Agarose XL beads provide:
- Purification of GST-tagged proteins (ca. 2 mg/ml binding capacity)
- High throughput protein purification: Large beads settle fast in 96 well plates
- Careful in-house production with high lot-to-lot reproducibility
PureCube GST Affinity resins compared to other suppliers
High protein binding capacity
In a comparative study with equivalent, market-leading resins, PureCube Glutathione Agarose XL proved to be a competitive alternative, exhibiting the same protein binding capacity. Figure 1 is an SDS-PAGE of GST purified on gravity columns packed with PureCube Glutathione Agarose XL and the equivalent resin from Competitor G. Both resins yielded up to 10 mg/mL protein. TL: total lysate; CL: cleared lysate; FT: flow-through; W1/W4: wash fractions; E1-E6: elution fractions.
Fig. 1: Purification yield is comparable to that of a market-leading resin. SDS-PAGE of GST expressed in E.coli and purified in gravity columns with PureCube Glutathione AgaroseXL and the equivalent resin from Competitor G. Up to 10 mg/ml protein yield was obtained with PureCube Glutathione Agarose XL(elution fractions E1-E6, Cube)
||Specific binding and purification of GST-tagged proteins
||Affinity to GST-tagged proteins
|High binding capacity
||2 mg / ml
||Delivered as a 50 % suspension
||300 - 500 µm
||Affinity agarose with GST-Antibody
- Lysis Buffer
- Wash Buffer
- Elution Buffer
- Ice bath
- Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
- 50 mL centrifuge tube
- Micropipettor and Micropipetting tips
- Disposable gravity flow columns with capped bottom outlet, 2 ml
- pH meter
- Optional: 15 mL conical propylene tubes pH meter
- UV/VIS Spectrophotometer
- End-over-end shaker
- SDS-PAGE buffers, reagents and equipment
- Optional: Western Blot reagents and equipment for control purposes using GST-Antibody.
|A.||Protein purification protocol|
- Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
- Resuspend the cell pellet in 10 mL Lysis Buffer and pour it into a 50 mL conical centrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption.
- Incubate on an end-over-end shaker at room temperature for 30 min, or at 4 °C for 1 h, depending on the temperature stability of the protein.
- Centrifuge the lysate at 10.000 x g for 30 min at 2-8 °C and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
- Resuspend the PureCube Glutathione Agarose XL by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50 % suspension (corresponding to 500 μL bed volume) into a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
- Add 5 mL of Wash Buffer and gently resupend the suspension to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant.
- Add the cleared lysate prepared in step 4 and incubate at 4 °C for 1 h on an end-over-end shaker. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
- Pour the complete suspension into a disposable gravity flow column with a capped bottom outlet.
- Remove the bottom cap of the column and collect the flow-through.
- Wash twice with 2.5 mL each of Wash Buffer.
- Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
- Elute the GST-tagged protein by adding 0.5 mL Elution Buffer.
- Repeat step 11 five times, for a total of six elutions. Collect each elution fraction separately. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
- Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
- Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
- Optional: Perform Western Blot experiment using GST Antibody.