Ion exchange technology of protein purification method


experimental scheme design

Unique autopure protein purification system

1. selection of gel medium
The choice of gel medium is mainly based on the molecular weight of the protein and impurity protein to be separated, and the gel with the corresponding separation range should be selected. At the same time, the resolution of
should be considered. Rate and stability factors. For example, if you want to separate the target protein from small molecular substances, you can choose Sephadex
according to the difference of their partition coefficient G-25 and G-50; For the desalination of small peptides and low molecular weight substances, Sephadex G-10, G-15 and bio-gelp-2 or P-4 can be selected< br> If it is a protein with a similar molecular weight, the gel with a maximum exclusion limit is slightly larger than that of the most high molecular weight substance in the sample. Application of specific gel filtration chromatography medium
As follows:

separation range of commonly used gel filtration chromatography media
gel mediumSeparation range of proteingel mediumSeparation range of protein
Sephadex G251~5Sepharose 2B70~40000
Sephadex G501.5~30Bio-Gel P-40.5~4
Sephadex G1004~150Bio-Gel P-105~17
Sephadex G2005~600Bio-Gel P-6030~70
Sepharose 6B10~4000Bio-Gel P-15050~150
Sepharose 4B60~20000Bio-Gel P-300100~400

2. pretreatment of gel medium


gel is fully swollen before application (glue: water =1:10), and the time of natural swelling is longer. The swelling can be accelerated by heating method, that is, boiling
. In water bath, the gel is heated to boiling, and 1~2h can achieve swelling. In the beaker, dry gel is added to water or buffer solution, stirred, placed quietly, and leached to the upper suspension,
Remove the gel fragments from the supernatant and repeat several times until the supernatant is cleared. p>

3. Selection of chromatographic column

The volume and height diameter ratio of the

column are closely related to the chromatographic separation effect. The volume, column length, column diameter and column ratio of the gel column bed must be determined according to the sample
. The quantity, nature and separation purpose were determined. For group separation, 2 ~ 30cm long chromatographic column is mostly used, and the column bed volume is 5
of the sample solution volume The column ratio is generally between 5 and 10; The fractionation generally requires about 100cm chromatographic column, and the column bed volume is required to be greater than the sample volume by 25
The column ratio is between 20 and 100

4. Packing of gel column

gel chromatography is different from other chromatographic methods. There is no force between solute molecules and stationary phase. The separation of sample components depends entirely on their respective flow
. Speed difference. When it is installed, close the post of the column, add some 1/3 column volume of water or buffer into the column, then stir the gel in the buffer along the side of the column
. Mix evenly and inject into the column slowly and continuously. When the gel is deposited about 5 cm, open the mouth of the column and control the flow rate at 1ml/min. p>

5. Sample handling and loading

According to the type of sample and purification analysis, appropriate buffer needs to be selected. In order to achieve good analysis effect, the sample loading volume must be kept at a small volume,
Generally, it is 1% ~ 5% of the column bed volume. The protein sample should be concentrated before loading, so that the sample concentration is not greater than 4% (the sample concentration has nothing to do with the partition coefficient),
However, it should be noted that for substances with large molecular weight, the solution viscosity will increase with the increase of concentration, limiting the molecular movement and affecting the flow rate. Before loading, the sample
Filter or centrifuge through the filter membrane to remove impurities that may block the chromatographic column

6. Elution and collection

The buffer solution of

gel filtration chromatography can be used as an eluent with a single buffer or a saline buffer. The main reasons are two aspects: solubility and stability of the protein. The buffer used shall ensure that the protein sample will not denature or precipitate. The pH shall be within the range of relatively stable and good solubility of the sample. At the same time, the buffer shall contain a certain salt (NaCl) to stabilize and protect the protein. During the elution process, always maintain a certain operating pressure, and the flow rate shall not be too high, which can be maintained at 0.5 ~ 3.0ml/min

Case introduction
Separation of protein
by gel filtration chromatography of Unique Autopure protein purification system Material
Chromatographic medium: Sephacryl S-200, protein separation range (5 ~ 250) × 103
Chromatographic column: xk16 / 60 pre installed column
Chromatographic equipment: unique autopure25
Mixed sample: containing monoclonal antibody with molecular weight of 180000; Bovine serum albumin (68000), lysozyme (14000)
NaOH 0.5 mol/L
NaCl 200 mmol/L
PB 20 mmol/L
PH7. 0 buffer
1. gel sterilization: after washing the columns with ultra pure water, rinse the column with 0.5mol/L NaOH, flow rate 3mL/min, rinse 3 column volume
2. Balance: after NaOH treatment, flush 2 column volumes with ultrapure water, and then flush 5 ~ 10 times the column volume with 7.0ph buffer containing 200mmol / lnacl and 20mmol / L Pb
3. Sample loading: after balancing, select the sample pump for sample loading. The sample loading flow rate is 3ml / min and the sample loading volume is 1ml
4. Elution: elute with equilibrium buffer after loading
5. Cleaning and storage
After purification, back flush 2 column volumes with 0.5mol/l NaOH for 30 ~ 60min. After flushing, forward flush with ultrapure water for 5
Column volume, wash 3 column volumes with 20% ethanol, then remove the column, seal both ends and store at low temperature

Problem analysis and solutions
1. How to purify samples before chromatographic separation
Before chromatographic separation, the sample shall be centrifuged and filtered. Centrifugation can remove most of the blocks. If the sample is still not clear after centrifugation, it can be filtered by filter membrane. The membrane made of cellulose acetate film or PVDF material can bind a small amount of protein nonspecifically

2. Incomplete solution exchange

Strictly control the sample loading volume, and the sample loading volume shall not exceed 30% of the column volume< br> If the sample solution has a large volume, it can be loaded several times. Pay attention to the time interval of each loading, and determine the next loading time according to the conductivity chromatographic peak

3. Low resolution

  • Improve the column loading quality and make the chromatographic column filled evenly

  • Increase the height of column bed

  • Control the sample loading volume, and the maximum sample loading volume shall not exceed 5% of the column bed volume

  • Control the viscosity of the sample to be consistent with that of the elution solution

  • Select the appropriate elution solution according to the characteristics of the sample, and adjust the ionic strength or hydrophilicity of the elution solution

  • choose the appropriate gel column (please refer to the above)

4. Poor symmetry of chromatographic peaks
 improve the quality of column installation and ensure uniform column installation - if the column installation is too loose, it is easy to cause tailing, and if the column installation is too tight, it will cause leading edge< br>  if the column is dirty, regenerate the chromatographic column
5. Causes and solutions of peak
 if the column bed is loose, reinstall the column or backwash the column
 the column sieve plate is blocked, and the sieve plate is cleaned by ultrasonic
 if the column is dry and cracked, reinstall the column