Purification Guide: Chromatography Resin Selection

For researchers, protein purification is a key step in studying the structure and function of proteins. Although there is no need to pursue high industrial-grade yield, the requirements for purity are extremely strict--structural research often requires ≥ 95%, and enzyme activity experiments must be at least ≥ 90%. Therefore, the first goal of purification is to be "pure enough", and on this basis, both yield and cost are taken into account.

The resolution, selectivity, and binding capacity of different resins determine the separation effect and also affect the success of your experiment. Affinity, ion exchange, hydrophobic interaction, multimodal, reversed-phase, gel filtration and other chromatography each has its own advantages and corresponds to different purification stages. The following table will help you quickly compare and find the most suitable match.

Table 1: Characteristics of different chromatography resins and their application in the purification stage

Among them, affinity chromatography has the best selectivity based on its ability to specifically bind to the target protein, but not to other impurity proteins. However, affinity chromatography is not optional for all protein purification, and it is not possible to produce an affinity chromatography resin for each target protein. There are many mature affinity chromatography resins for widely used protein purification tags, such as His, Fc, GST tags, etc. Affinity chromatography is the best choice for protein purification in scientific research field. For some proteins which have low expression level and are difficult to purify, more than one purification tag can be added during expression, and two-step affinity chromatography can be used.

When using affinity chromatography, it is necessary to understand the principle of affinity. According to the factors that may affect the purification effect, the sample adjustment, buffer selection, chromatography steps and other aspects of the chromatography process can be optimized. As a common example, the His-tagged protein helps us better understand how affinity chromatography works in practice.

The imidazole group on the side chain of histidine (His) can form coordination bonds with various metal ions (such as Ni^{2 +}, Co^{2 +}, Cu^{2 +}, Zn^{2 +}, etc.) and selectively bind. Among them, Ni^{2 +} is the preferred metal ion for purifying His tag protein, and it is also the most widely used metal ion. At present, tetravalent chelated NTA affinity chromatography resins and pentavalent chelated TED are widely used. Among them, the Ni ions chelated in TED form are extremely stable, can tolerate reducing agents and chelating agents such as EDTA, and are suitable for mammalian cell expression supernatants, direct loading and purification and renaturation solution loading with reducing agent. When selecting other metal ions for purification, IMAC resin can be selected to chelate the metal ions by itself and then purify them.

Table 2: Introduction to the characteristics of resins in different chelated forms of BioLink

When using Ni affinity, pay attention to: metal ions are positively charged. In order to avoid non-specific binding, a certain concentration of NaCl should be added to the buffer; At the same time, low concentration imidazole can be added to inhibit non-specific adsorption dominated by non-His tag. After the sample is loaded and combined, an appropriate concentration of imidazole should also be selected as Wash to remove the impurity protein with weak non-specific binding and increase the concentration of the target protein in the eluate.

Case 1: Purification of Recombinant Collagen

Sample: Recombinant Collagen

Column: Ni Chromstar^® Excel (0.77 * 10 cm, column volume (CV) = 4.7 ml)

Buffer A:  20 mM PB + 0.15 M NaCl (pH 7.5)

Buffer B: 20 mM PB + 0.15 M NaCl (pH 7.5) + 0.5 M Imidazole (pH 7.5)

Table 3: Detailed steps of recombinant collagen purification

Figures 1 and 2: Ni resin chromatogram and gel diagram

Table 4: Common problems in protein purification

If purity is not sufficient after affinity purification, polishing may be followed by ion exchange, hydrophobic interaction, multimodal or gel filtration chromatography.

Select the chromatography method based on the difference in the properties of the target protein and the impurity protein:

• Ion Exchange Chromatography: Large difference in isoelectric points

• Hydrophobic Interaction Chromatography: Large differences in hydrophobicity

• Gel Filtration Chromatography: Large difference in molecular weight

• Multimodal Chromatography: Ion exchange and hydrophobic interaction chromatography alone cannot effectively separate

• Reversed-phase Chromatography: Generally not selected in protein purification, but can be selected in the purification of small molecule polypeptides

Among them, ion exchange chromatography is convenient to operate and widely used. It is the preferred purification method besides affinity. Especially when the differences in protein properties are not fully known, it can be used as the first choice for testing. Differences in molecular weight can usually be found from SDS PAGE results, and tagged protein fragment impurities are also common in affinity samples.

Therefore, gel filtration chromatography is also widely used in scientific research. All purification methods except gel filtration chromatography involve changes in pH or conductivity conditions. When selecting a chromatography method, the stability of the target protein should be considered first, and the optional chromatography method and operating range should be determined.

Table 5: BioLink provides a variety of chromatography resins and pre-packed columns.

The products marked in bold are suitable for applications in scientific research fields.

Taking ion exchange as an example, when the target protein has a net positive charge (the solution pH is lower than PI), it can be combined with cation exchange chromatography resin (S, SP, CM); On the contrary, it can be combined with anion exchange chromatography resin (Q, DEAE) to select appropriate pH conditions and corresponding chromatography resin according to the stable pH range of the protein and maximizing the charge difference between the target protein and the impurity protein. Samples need to bind at low conductance and elute at increasing salt concentration or changing pH. In the initial experiment, after protein binding, it can gradually transition from low conductivity solution to high conductivity solution in a linear gradient manner, so as to improve the resolution and find suitable conditions for removing impurities and eluting the target protein.

Case 2: SP Chromstar^® FF Purified Collagen Case

Sample: Type I Collagen

Molecular Weight: 300-400 kDa

Isoelectric Point: 7-9 (theoretical)

Source: CHO cell expression

Equilibration: 50 mM HAc-NaAc, pH 4.0, Cond 7.0 mS/cm

Elution: 50 mM HAc-NaAc+1 M NaCl, pH 4.0

RT=5 min

Figure 3: Type I collagen chromatography

Gel filtration chromatography resins separate proteins based on molecular weight differences and are usually not affected by solution conditions. However, there is a weak ion exchange between purely used gel filtration chromatography resins and proteins. Generally, a certain concentration of salt is added to samples and solutions for shielding. In order to achieve better resolution, the loading volume is generally limited to within 5% of the column volume.

Case 3: Geldex^® 200 PG separates proteins with different molecular weights

Column: 16/600 pre-packed column (Geldex^® 200 PG)

Sample:

1. Myoglobin, 5.4 mg/mL, Mr 17 000

2. Ovalbumin, 36 mg/mL, Mr 44 000

3. HSA, 36 mg/mL, Mr 66 000

4. IgG, 2.8 mg/mL, Mr 158 000

5. Equine spleen ferritin, 3.4 mg/mL, Mr 440 000

Loading Amount: 500 μL

Flow Rate: 1 mL/min (30 cm/h)

Buffer: 10 mM PB, 140 mM NaCl, 2.7 mM KCl, pH 7.4 (PBS)

Figure 4: Protein chromatography performance of Geldex^® 200 PG for different molecular weights

The starting conditions and ending conditions of different chromatography modes are different. Puredex^® G25M can be selected for solution exchange (including desalting) of samples or final products between different chromatography modes. Its protein molecular weight is much larger than that of various salt ions, so compared with gel filtration chromatography resins for polishing, such as Geldex^® 200PG, the loading amount can reach 25% of the column volume during desalination.

Table 6: Starting and ending conditions for different chromatography modes

Case 4: Puredex^® G-25M Desalination

Resin: Puredex^® G-25M

Column: Chrom-LinX^® 26/400 (H = 34 cm), column volume 170 ml

Sample: A protein sample (~ 750 kd)

Sample Volume: 20 ml, 12% column volume

Flow Rate: 8 mL/min, 90 cm/h

Buffer: 10 mM acetic acid-sodium acetate, pH 6.0

Figure 5: Chromatogram of a protein sample

To sum up, affinity chromatography is the first choice for protein purification in scientific research fields, and ion exchange chromatography and gel filtration chromatography also have many applications. There is no fixed paradigm for the purification process, and a variety of chromatography modes need to be selected for combination according to the target protein and impurities. Space is limited and the details of each chromatography cannot be detailed. If necessary, please contact the corresponding sales and technology. BioLink will provide technical and product support at any time.