Mbiotech Products > Glutathione Excellose® Kit

The Glutathione Excellose® Kit is designed for rapid single step purification of recombinant derivatives of glutathione S-transferase, other glutathione S-transferase or glutathione dependent proteins. It consists of glutathione Excellose resin, microcentrifuge tubes, spincolumn, collection tubes, and various buffers. Glutathione Excellose® is a affinity chromatography medium used for simple and rapid purification of GST (glutahione S-transferase) fusion proteins. This purification system is based on the remarkable selectivity of Glutathione resin for recombinant proteins carrying a affinity tag, the GST tag. Proteins are quickly and easily purified to near homogeneity with microcentrifuge. The gentle elution (10 mM reduced glutathione) avoids target protein denaturation. The Glutathione Excellose® Kit is ideal for screening, analysis of expression, and purification prior to scaling up.

The kit offers a number of advantages:

- Simple : One-step purification of multiple

sample with microcentrifuge.

- Flexible : The resins are compatible with variety

of common buffer.

- Efficient : Binding capacity is up to 75 μg of GST fusion protein per

spin column (final vol. of resin = 250 μl)

- Convenient : Complete system that includes all

necessary components

Kit Contents

Components Amount(21020) (21050) Storage

Glutathione Excellose® 10ml 27ml 2-8°C

Spin Column 20 ea 52 ea Room Temperature

Collection Tube 20 ea 52 ea Room Temperature

10x washing buffer 60 ml 60 ml 2-8°C

1x Elution buffer 60 ml 120 ml 2-8°C

Protocol 1 ea

Instrument and lab supplies

Table top centrifuge, 1.5 ml microtubes, microtube rack

Buffers

Washing Buffer : PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,

2 mM KH2PO4), pH 7.4

Elution Buffer : washing buffer + 10 mM reduced glutathione

Storage conditions

All components are guaranteed for 12 months from the date of purchase when

stored at 2-8°C and used as described in this manual.

HOW TO USE

Glutathione Excellose® kit contains Glutathione Excellose®,
spin column, collectionTube, and stock solution.

Protocols

Culture Volume: 1-10 ml

Min. Elution Vol.: 200 μl

Before cell homogenization, check the protein characteristics (soluble or insoluble

proteins) and protein expression level.

If the protein expression level is high (> 10 mg / liter), the original culture medium must be 5 times more concentrated for sonication. In other words, if the protein to be purified is soluble with a high expression level, 10ml of culture must be resuspended in 2ml of buffer* for sonication.

If the protein expression level is low ( 2 ~ 5 mg/liter), the original culture medium

must be 20 times concentrated.

* When applying the purification conditions provided in this manual, make it sure to use the washing buffer described in this manual. If the protein solution to be purified is different from the washing buffer used in this manual,

1) Replace the protein solution with the washing buffer of this manual or

2) Change the washing buffer to match with the protein solution.

For example;

Protein solution – when it’s 50 mM sodium acetate, 0.3 M NaCl, pH 6.0

-1) Prepare a new cell culture in the buffer system of this manual or replace the buffer by using dialysis.

Protein solution : PBS, , pH 7.4

Washing buffer : 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4

Elution buffer : Washing buffer + 10 mM reduced glutathione

-2) Use the same buffer as protein solution for washing buffer.

Washing buffer : 50 mM sodium acetate, 0.3 M NaCl, pH 6.0

Elution buffer : Washing buffer + 10 mM reduced glutathione

Note: Save 30 μl of each fraction for SDS-PAGE analysis.

1. Resuspend the cell pellet from 1-10 ml culture in 1 - 3 ml Washing buffer.

2. Sonicate or homogenize cells by sonicator or homogenizer.

Spin for 10 minutes at 12,000 x g.

3. Collect the cleared lysate.

4. Put the Column with Collection Tube to the microtube rack.

Transfer 500 μl

5. Vortex the Glutathione Excellose® tube and pipet 500 μl of the slurry to Column(final resin vol. 250μl).

6. Centrifuge at 700 x g (approx. 2,000 rpm) for 2 min.

7. Discard the eluate in the Collection Tube.

Add 500 μl of Washing Buffer in the Column and centrifuge.

8. Repeat step 7.

9. Discard the eluate, load up to 500 μl of cell lysate and store it at normal room temperature for 2 minutes, and separate it with a centrifuge at 2,000 rpm for 2 min.

- For protein solution with a low expression level, the purification yield can be improved by repeating step 9.

- The centrifugation speed is very important. If the speed is too high, it can result in low binding capacity because there will be less time for the protein solution and resin to bind each other.

- It may take more time for centrifugal separation depending on the concentration or viscosity of protein solution.

10. Collect the flow-through fraction.

11. Repeat step 9 – 10, if necessary.

12. Add 500 μl of Washing Buffer and centrifuge.

- If the protein expression level is high, washing it twice should be enough. But if the protein expression level is low, the washing process must be repeated 3 times to produce highly purified proteins.

13. Collect the wash fraction

14. Repeat step 12 – 13, if necessary.

15. To elute MBP fusion protein, add 300 μl Elution Buffer to the column and incubate the sample for 3 minutes at room temperature. Centrifuge for 2 minutes at 2,000 rpm.

16. Collect the eluate

17.Analyze the purification yield by using SDS-PAGE.

Use SDS-PAGE to analyze the purification yield in the order of:

protein solution, flow-through fraction①, wash fraction② and elution fraction③.

Above is one of the common protein purification methods, which may vary depending on the protein binding characteristics of each user.

ORDERING INFORMATION

Catalog No	Package
21020	20 columns/pkg
21050	50 columns/pkg

FIGURE

GST fusion protein purification
E.coli lysate containing GST fusion protein was loaded to Glutathione Excellose® spin column.