Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample. An example is if you estimate the aggregate/dimer content in your antibody sample by size exclusion chromatography.
Preparative chromatography is used to purify sufficient quantities of a substance for further use, such as for functional or structural studies.
To learn more, download our free handbooks about protein purification
For sample preparation before chromatography, you should select the filter pore size based on the bead size of the chromatographic medium as shown in the table below.
| Particle size of chromatographic medium | Nominal filter pore size |
|---|---|
| 90 µm or greater | 1 µm |
| 30 or 34 µm | 0.45 µm |
| 3 µm, 10 µm, 15 µm, or when extra-clean samples or sterile filtration is required. | 0.22 µm |
Learn more about our range of Whatman products for laboratory filtration.
We recommend you to use the
Purify app
– our protein purification media selection app. It’ll help you to quickly select the right product(s) for your needs. Based on your answers to a few intuitive questions it’ll guide you to a recommended product. The
Purify app
will also provide a summary of applications, important technical data, and useful hints regarding both system and column compatibility for the product.
If the sample preparation protocol is followed, and recommended buffers are used as stated in the HiTrap protocol, you should be able to use a HiTrap column at least 5 times. Please note that HiTrap Streptavidin HP columns can only be used once.
Learn more about our HiTrap collection.
The static binding capacity (SBC, also called total protein capacity) is normally measured in batch mode in a beaker. It’s usually referred to as the maximum amount of protein bound to a chromatography medium at given solvent and protein concentration conditions. SBC varies considerably depending on the protein loaded.
Dynamic binding capacity (DBC), on the other hand, is the binding capacity under operating conditions (i.e., in the packed affinity chromatography column during the sample application and washing procedures). As the DBC reflects the impact of mass transfer limitations that may occur as flow rate is increased, it is much more useful in predicting real process performance than a simple determination of saturated or static capacity.
Linear flow rates are measured in units of distance traveled per time (cm/min). The linear flow rate is independent of the diameter of the column. Volumetric flow rates are in units of volume per time (ml/min)..
To convert between linear flow and volumetric flow rate you can use our flow rate converter.
A chromatography system should be used when reproducible results are important and when manual purification becomes too time-consuming and inefficient..
You should choose your purification method based on the requirements of your application (e.g., sample volume, purity, amount of protein required, presence of toxic components, number of samples to be processed) and on what equipment and experienced are available in your laboratory.
Discover our ÄKTA lab-scale chromatography systems.
| Cause | Remedy |
|---|---|
| Protein may have been degraded by proteases. | Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteinases. |
| Protein adsorption to filter during sample preparation. | Use a filter of a different membrane type. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and are good starting points for determining the most suitable membrane type for your application. Learn more about our range of Whatman filters. |
Antibody Purification
Protein G is a good first choice for general purpose capture of antibodies at laboratory scale, as it binds to IgGs from a broader range of eukaryotic species than protein A. Protein G also binds to more subclasses of IgG than protein A. For details on the relative binding strengths of antibodies from various species to protein G and protein A, see page 48 of our Antibody Purification handbook.
As the elution step in antibody purification often requires lowering the pH, there is a risk that sensitive antibodies can lose their activity. To prevent this, there are some general recommendations that may help you:
- Prepare collection tubes with 60 to 200 µl of 1 M Tris-HCl buffer pH 9.0 per milliliter of eluate. This may preserve antibody activity as the final pH of the eluate will be approximately neutral.
- Determine the highest pH that preserves the activity of your antibody when you optimize your elution conditions.
- Desalt and/or transfer your eluate to a suitable buffer directly after elution for quick pH neutralization of the eluted antibody.
Protein L binds immunoglobulins through interaction with the immunglobulin light chain. It was first isolated from the surface of the bacterial species Peptostreptococcus magnus, and named protein L for light chain. No part of the heavy chain is involved in the interaction, which means that protein L binds a wider range of antibody classes than protein A or protein G.
Our Capto L affinity chromatography medium (resin) binds specifically to the variable region of the antibody kappa light chain. The Capto L resin is designed for one-step purification of a wide range of antibody fragments such as Fabs, single chain variable fragments, and domain antibodies.
| Cause | Remedy |
|---|---|
| Protein may have been degraded by proteases. | Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteinases. |
| Protein adsorption to filter during sample preparation. | Use a filter of a different membrane type. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and are good starting points for determining the most suitable membrane type for your application. Learn more about our range of Whatman filters. |
Tagged Protein Purification
Sometimes you're not particularly interested in purifying proteins. You just want to get it over and done with so you can get on with the experiments that will help you understand its role. This is where tagging your protein with something that adds biospecific affinity comes in handy. It allows you to simplify the purification protocol greatly, sometimes to the extent that you can use a standard protocol.
Remember though, that tagging may not always be the right solution as adding a tag can introduce changes compared to the native protein, leading to undesirable effects. For example, if you are interested in functional studies, a tag may introduce uncertainty, or worse, alter or totally destroy the function of your target protein.
In addition, the tag itself can interfere with your ability to use the protein the way you want. This is of particular importance when proteins are to be used as therapeutics and you want to be as close to the native form as possible. In most situations however, tags, especially small ones like a His-tag, have no negative effects in terms of biophysical characterization.
| Advantages of using a tag | Disadvantages of using a tag |
|---|---|
| Simple, generic purification using affinity chromatography (AC) as a first step. |
Tags may interfere with protein structure and affect folding and biological activity. |
| Tags are easy to detect, which allows for generic detection methods. |
If the tag needs to be removed, cleavage may not always be 100% successful, and sometimes some amino acids may be left. |
| Some tags can improve protein solubility and stability. |
Only some tags can be used under denaturing conditions. |
To learn more, download our Recombinant Protein Purification handbook.
With the introduction of affinity tagging of proteins, protein purification was dramatically simplified. It was now possible to use generic protocols, which enabled much more efficient and easy protein purification. This led to tags that do more than just purify protein, such as improving the solubility or stability.
Below you find some characteristics of the tags that are most commonly used in protein purification today.
| His | Strep-tag™ II | GST | MBP | FLAG™ | |
|---|---|---|---|---|---|
| Size | 6 aa | 8 aa | 26 kDa | 40 kDa | 8 aa |
| Protein binding capacity | 40 mg/ml | 6 mg/ml | 30 mg/ml | 10 mg/ml | 0.6 mg/ml |
| Purity | + | +++ | ++ | ++ | +++ |
| Increased solubility | No | No | Yes | Yes | No |
| Risk for interference with function | Low | Low | High | High | Low |
To learn more, download our Recombinant Protein Purification handbook.
The “Classic” His-tag
The polyhistidine tag is by far the most commonly used tag for protein purification today. The reason for this is simple—it’s so small that it’s unlikely to interfere with the structure or function of your target protein, and you usually don’t have to remove it before using the purified protein in your assays (except for e.g., structure determination using X-ray crystallography).
Another great benefit of the histidine tag is that purification is quite straightforward, and that there is a great selection of ready-to-use purification products in a multitude of formats available to choose from. Different chromatography media are available that will provide different trade-offs between recovery, capacity, and purity.
Adding a histidine tag means that you typically add 4-6, sometimes up to 10, histidine residues to either the N- or the C-terminal of your protein. The aromatic group of the histidine residues binds to chelated divalent metal ions. Nickel ions are the most commonly used, but cobalt, zinc, and copper ions can also be used. Zinc ions are the best choice for the environment. Regardless of the ion, imidazole is always used for elution.
The main drawback of the polyhistidine tag is that it often requires some optimization of the imidazole concentration in your sample and in the buffers for column equilibration and washing in order to minimize binding of host cell proteins with high histidine content.
Strep-tag™ II
Strep-tag™ II is a peptide tag that binds very specifically to Strep-Tactin™, a modified version of streptavidin. Being small, it shares the benefits of the His-tag, and adds significant improvement in the purity you can expect.
In addition to the chromatography media being more expensive and having much lower binding capacity than media for purifying his-tagged proteins, the agent used for elution, desthiobiotin, is more expensive than imidazole.
GST for Purity and Solubility
Another very common tag is the enzyme glutathione-S-transferase (GST). It binds specifically to glutathione immobilized on chromatography media, and therefore often gives very high purity. Another benefit is that it can increase the solubility of the protein it is fused to. However, being big (26 kDa), it often needs to be cleaved off in order to eliminate interference with the structure and function of your target protein.
While there are GST chromatography media with high binding capacity, the binding kinetics are slow. You therefore have to load your sample at low flow rates.
MBP tag
Maltose Binding Protein (MBP) is another protein tag that can be used for purification as an alternative to GST. Whilst providing the same benefits of high specificity and ability to improve solubility of your protein, it is larger than GST (40 kDa), so it typically needs to be removed before you can use your protein. Lower binding capacity compared to GST and a more limited number of purification products available make this tag a secondary choice to GST.
FLAG™ tag
If none of the tags discussed above work, the FLAG™ peptide-tag is a small tag that binds very specifically to a specific antibody currently only available on one type of chromatography medium. In addition to the high specificity and thereby purity that can be expected, another benefit of this tag is that it is small, and therefore unlikely to interfere with the function of the protein it is fused to.
The main drawback is that the affinity medium is based on an immobilized antibody, and therefore has limited binding capacity, resulting in either large column sizes or small amounts of purified protein per batch. The chromatography medium also comes at a higher cost than alternative affinity media.
To learn more, download our Recombinant Protein Purification handbook.
Yes you can, but removing the tag is not always straightforward. Tag removal always includes using a sequence-specific protease. The blood clotting factors thrombin and factor Xa are most commonly used, but you need to make sure that their respective cleavage sequences (LVPR↓GS and IEGR↓) are not present within your target protein.
Some tags, such as the glutathione-S-transferase (GST)-tag can be removed with more specific proteases, such as our PreScission protease (human rhinovirus 3C protease, cleavage sequence LEVLFQ↓GP), and this is unlikely to affect your target protein.
Affinity chromatography in general
| Cause | Remedy |
|---|---|
| Protein may have been degraded by proteases. | Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteinases. |
| Protein adsorption to filter during sample preparation. | Use a filter of a different membrane type. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and are good starting points for determining the most suitable membrane type for your application. Learn more about our range of Whatman filters. |
| Sample has precipitated. | May be caused by removal of salts or unsuitable buffer conditions. |
| Hydrophobic proteins. Protein is still attached to ligand. | Use chaotropic agents, polarity reducing agents or detergents. |
His-tagged proteins
| Cause | Remedy |
|---|---|
| No histidine-tagged protein present in the starting material. | Verify presence of histidine-tagged protein in the starting material, e.g., by Western blotting. |
| Elution conditions are too mild (histidine-tagged protein still bound). | Elute with increasing imidazole concentration or decreasing pH. |
| Protein has precipitated in the column. | Decrease amount of sample, or decrease protein concentration by eluting with linear imidazole gradient instead of imidazole steps. Try detergents or change NaCl concentration. |
| Nonspecific hydrophobic or other interactions are occurring. | Add a nonionic detergent to the elution buffer (e.g., 0.2% Triton X-100) or change the NaCl concentration. |
| Histidine-tagged protein is not completely eluted. | Elute with a larger volume of elution buffer and/or increase the concentration of imidazole. |
| Nickel ions are being stripped from the matrix. | Change to Ni Sepharose excel if the sample causes stripping, e.g., if the histidine-tagged proteins are secreted into eukaryotic cell culture supernatants. |
Strep-tag™ II proteins
| Cause | Remedy |
|---|---|
| Protein is found in the flowthrough. | Buffer/sample composition is not optimal. Check the pH and composition of the sample and binding buffer. pH should in general be pH 7 or higher. |
| Strep-tag™ II is not present. | Use protease-deficient E. coli expression strains. Add protease inhibitors during cell lysis. |
| Strep-tag™ II is not accessible. | Fuse Strep-tag™ II to the other protein terminus. Use another linker. |
| The ligand is blocked by biotinylated proteins from the extract. | Add avidin if biotin containing extracts are to be purified. The biotin content of the soluble part of the total E. coli cell lysate is about 1 nmol per liter of culture (A550 = 1.0). Add 2 to 3 nmol of avidin monomer per nmol of biotin. |
GST-tagged proteins
| Cause | Remedy |
|---|---|
| The volume of elution is insufficient. | Increase the volume of elution buffer used. In some cases, especially after on-column cleavage of a tagged protein, a larger volume of buffer may be necessary to elute the tagged protein. |
| The time allowed for elution is insufficient. | Increase the time used for elution by decreasing the flow rate during elution. With GSTrap columns, for best results use a flow rate of 0.2 to 1 ml/min (1 ml HiTrap column) and 0.5 to 5 ml/min (5 ml HiTrap column) during sample application. For centrifugation methods, decrease the centrifugation speed during elution. |
| The concentration of glutathione is insufficient. | Increase the concentration of glutathione in the elution buffer: The 10 mM recommended in this protocol should be sufficient for most applications, but exceptions exist. Try 50 mM Tris-HCl, 20 to 40 mM reduced glutathione, pH 8.0 as elution buffer. |
| The pH of the elution buffer is too low. | Increase the pH of the elution buffer. Increasing the pH of the elution buffer to pH 8 to 9 may improve elution without requiring an increase in the concentration of glutathione used for elution. |
| The ionic strength of the elution buffer is too low. | Increase the ionic strength of the elution buffer. Adding 0.1 to 0.2 M NaCl to the elution buffer may also improve results. |
| The glutathione in the elution buffer is oxidized. | Use fresh elution buffer. Add DTT. |
| Nonspecific hydrophobic interactions cause nonspecific interaction with the medium or aggregation of proteins, preventing solubilization and elution of tagged proteins. | Add a nonionic detergent to the elution buffer. Adding 0.1% Triton X-100 or 2% n-octylglucoside can significantly improve elution of some GST-tagged proteins. |
MBP-tagged proteins
| Cause | Remedy |
|---|---|
| Factors in the crude extract interfere with binding. | Include glucose in the growth medium to suppress amylase expression. |
| MBP-tag is not present. | Use protease-deficient E. coli expression strains. Add protease inhibitors during cell lysis. |
| MBP-tag is not accessible. | Fuse the MBP-tag to the other protein terminus. Use another linker. |
| Protein has precipitated in the column due to high protein concentration. | Clean the column according to instructions. In the following run decrease the amount of sample, or decrease protein concentration by eluting with a linear gradient instead of step-wise elution. Try detergents or change the NaCl concentration. If an MBPTrap HP 1 ml column has been used, change to the larger MBPTrap HP 5 ml. This will reduce the final concentration, provided that the same amount of sample is applied.For quick scale-up, connect two or more columns in series by screwing the end of one column into the top of the next. Note, however, that connecting columns in series will increase back pressure. |
To learn more, download our Recombinant Protein Purification handbook.
Purification of Untagged Proteins
Untagged proteins may be purified with a single affinity step if there is a ligand that binds the protein biospecifically. If it’s not possible to use an affinity step, you probably need to use a multistep purification strategy.
Multistep purification involves choosing the right combination of purification techniques, based on the properties of your target protein. Valuable information on how to plan and perform protein purification can be found in our Strategies for Protein Purification handbook.
If you are interested in functional studies, a tag may introduce uncertainty, or worse, alter or totally destroy the function of your target protein. In addition, the tag itself can interfere with your ability to use the protein the way you want. This is of particular importance when proteins are to be used as therapeutics and you want to be as close to the native form as possible.
| Cause | Remedy |
|---|---|
| Protein may have been degraded by proteases. | Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteinases. |
| Protein adsorption to filter during sample preparation. | Use a filter of a different membrane type. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and are good starting points for determining the most suitable membrane type for your application. Learn more about our range of Whatman filters. |
Size Exclusion Chromatography (SEC)
Many factors can influence the resolution in size exclusion chromatography (SEC), such as the particle size of the medium, the column length, and the sample volume.
But another aspect that might be less obvious is the contribution of the liquid chromatography (LC) system itself. It is important to minimize the dead volumes in the system, for example by using short and narrow tubing and removing all unnecessary components in the system flow path. For more details, see page 18-19 of our Size Exclusion Chromatography handbook.
The pressure over the packed bed depends on several parameters including:
- Flow rate
- Viscosity of sample and eluent
- Running temperature
- Chromatography medium particle properties
- Column packing
The packed bed is best protected by controlling the flow rate. To prevent too high back pressure when using samples and/or buffers with high viscosity or when running at low temperature (+ 4°C to + 8°C), you should decrease the flow rate by approximately 50% compared to the recommended flow rate.
Clogged filters can also increase back pressure. Check that the inline filter of your system is not clogged. In columns where you can change the top filter you should also check that the top filter is not clogged.
We suggest that you also make a routine of regularly performing a CIP (cleaning-in-place) of your column. A suitable protocol is available in the instruction for use for each of our columns, which you can find on the product page. Product pages for SEC columns are found by clicking through from our size exclusion chromatography category page.
The system setup has impact when measuring the efficiency of the column (HETP). If you do not have the exact system setup as was used to determine the HETP at our production facility you will get a different value than in the certificate of analysis (CofA). To follow the performance of each column over time, we recommend that you perform your own initial efficiency test.
Depending on the column type, you might be able to “rescue” the column. If it is a column that can be opened and repacked you should turn down the adapter to the medium bed. After this you should test the column efficiency to check the performance. If the performance is acceptable, you can continue to use the column. If not, repack the column if possible. In other cases you will need a new column.
Small amounts of air will normally not affect the performance of the column. You can remove small air bubbles by passing degassed buffer through the column. Make sure that your buffers, system, and columns all have the same temperature, especially if your buffers have been stored in a fridge or a cold room.
When connecting your SEC column to the system, make sure that the all parts of the system (including tubing) is filled with liquid to prevent air from entering the system.
All our SEC columns are packed together with an instruction for use with information about that particular column’s chemical stability, sometimes called “buffers and solvent resistance”. The chemicals in those lists have been tested with the column in question.hromatography is used to purify sufficient quantities of a substance for further use, such as for functional or structural studies.
If you’ve lost your hardcopy instruction for use you can find a pdf version on the product page. Product pages for SEC columns are found by clicking through from our size exclusion chromatography category page.
| Cause | Remedy |
|---|---|
| Protein may have been degraded by proteases. | Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteinases. |
| Protein adsorption to filter during sample preparation. | Use a filter of a different membrane type. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and are good starting points for determining the most suitable membrane type for your application. Learn more about our range of Whatman filters. |
| Sample has precipitated. | Can be caused by removal of salts or unsuitable buffer conditions. |
| Non-specific interactions between protein and matrix. | Increase salt concentration in the buffer, up to 300 mM NaCl. |
| Ionic interactions between protein and matrix. | Maintain ionic strength of buffers above 50 mM (preferably include up to 300 mM NaCl). |
| Hydrophobic interactions between protein and matrix. | Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent, e.g. 5% isopropanol. |
For more troubleshooting information, download our Size Exclusion Chromatography handbook.
Ion Exchange Chromatography (IEX)
Ion exchange chromatography (IEX) separates molecules on the basis of differences in their net surface charge. IEX can be used for purification of proteins, peptides, nucleic acids, and other charged biomolecules, offering high resolution and group separations with high loading capacity..
Ion exchange chromatography can separate molecular species that have only minor differences in their charge properties, for example two proteins differing by one charged amino acid. IEX is well suited for capture, intermediate purification, or polishing steps in a purification protocol.
To learn more, download our Ion Exchange Chromatography handbook.
Start with a strong ion exchanger. If the selectivity is not good enough, try a weak ion exchanger. Strong ion exchangers (Q, SP, and S) have constant capacity over a wide pH range, while the capacity of weak ion exchangers (DEAE, ANX, and CM) varies with pH.
Proteins can be purified with both cations and anions but one may be more efficient for your particular protein. If you know the isoelectric point (pI) of your protein, start with a cation exchanger for your capture step if pI>7, and with an anion exchanger if pI<7.
Buffering ions should have the same charge as the functional groups on the IEX medium.
- For anion exchange, choose a buffer 0.5-1 pH units above isoelectric point (pl), as proteins will bind tighter with increasing pl.
- For cation exchange, choose a buffer 0.5-1 pH units below pl, as proteins will bind tighter with decreasing pl.
For both anion and cation exchange, you need to consider the stability window of your protein, as many proteins are unstable when the pH is close to the isoelectric point (pI) of the protein.
Tables with pH intervals for buffering substances are found in our Ion Exchange Columns and Media Selection Guide.
| Cause | Remedy |
|---|---|
| Protein may have been degraded by proteases. | Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteinases. |
| Protein adsorption to filter during sample preparation. | Use a filter of a different membrane type. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and are good starting points for determining the most suitable membrane type for your application. Learn more about our range of Whatman filters. |
| Sample has precipitated. | Check salt conditions, adjust to improve sample solubility. |
| Hydrophobic interactions between protein and matrix. | Add denaturing agents, polarity reducing agents, or detergents. Add 10% ethylene glycol to running buffer to prevent hydrophobic interactions. |
| Non-specific adsorption of protein to matrix. | Reduce salt concentration to minimize hydrophobic interaction. Add suitable detergent or organic solvent, e.g. 5% isopropanol. If necessary, add 10% ethylene glycol to running buffer to prevent hydrophobic interactions. |
For more troubleshooting information, download our Ion Exchange Chromatography handbook.
Sample Preparation/Desalting
Use desalting/buffer exchange when needed:
- Before purification.
- Between purification steps.
- After purification.
Desalting and buffer exchange can take less than 5 minutes per sample with greater than 95% recovery for most proteins.
When desalting, sample volumes of up to 30% of the total volume of the desalting column can be processed. Moreover, the high speed and capacity of the separation allows even relatively large sample volumes to be processed rapidly and efficiently. Desalting therefore provides several advantages over dialysis.
To prevent possible ionic interactions, the presence of a low salt concentration (25 mM sodium chloride) is recommended during desalting and in the final sample buffer. Volatile buffers such as 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate can be used if it is necessary to avoid the presence of sodium chloride.
- The sample should be fully dissolved. Centrifuge or filter your sample to remove particulate material.
- Always use degassed buffers to avoid introducing air into the column.
- Sample concentration up to 70 mg/ml protein should not influence the separation when using normal aqueous buffers.
Desalting columns are used not only to remove low molecular weight contaminants such as salt, but also for buffer exchange before and after different chromatography techniques and for the rapid removal of reagents to terminate a reaction. Examples of group separations include:
- Removal of phenol red from culture fluids prior to anion exchange chromatography or nucleic acid preparations.
- Removal of unincorporated nucleotides during DNA sequencing.
- Removal of free low molecular weight labels.
- Termination of reactions between macromolecules and low molecular weight reactants.
- Removal of products, cofactors, or inhibitors from enzymes.
- Removal of unreacted radiolabels such as adenosine triphosphate (ATP) from nucleic acid labeling reactions.