Protein Purification Support—Getting Started

Please view our selection chart to choose the right immobilization solution for your experiment.

Please see the table below.

AminoLink™ and AminoLink™ Plus Supports are activated with an aldehyde group to form a Schiff base, which is reduced to a stable, non-reversible secondary amine. Coupling efficiency often exceeds 85% with this support.

AminoLink™ Plus Coupling Resin is activated at a higher level and has higher flow rates than the original AminoLink™ Coupling Resin, resulting in higher capacity and faster purification. AminoLink™ Supports can be used to immobilize any molecule with a primary amine.

NHS- activated agarose reacts with primary and secondary amines to form stable amide and imide linkages respectively. Most proteins can be coupled in 30 mins with greater than 85% coupling efficiency. No hazardous chemicals are used, and the agarose is adaptable to use with spin columns, batch methods, or FPLC. The resin is resusable typically up to 10 purifications with the immobilized ligand, and has a high binding capacity (>30 mg/mL slurry or >25 mg/mL dry resin).

Both react with primary amines, but AminoLink™ Coupling Resin is a two-step conjugation. First a Schiff base is formed between the amine and the aldehyde on the resin, which is then reduced to a stable secondary amine with sodium cyanoborohydride, whereas NHS-Activated Agarose utilizes an NHS ester to form an amide bond in a one-step conjugation.

Iodoacetyl-derivatized support reacts with free sulfhydryl groups on a protein/peptide or other biological molecule, resulting in a stable thioether bond (non-reducible). This support is ideal for peptides with a terminal cysteine or for orienting monoclonal antibodies that have amines in the antigen-binding site.

The benefits include high-capacity immobilization of 1–10 mg of oxidized antibody or other glycoprotein per milliliter of resin. Immobilization is fast (in as few as 30 mins) and can achieve at least 90% coupling of most glycoproteins in less than 4 hours. The hydrazide-activated UltraLink™ resin conjugates only to purified glycoproteins whose sugar groups have been gently oxidized with periodate.

No stabilization step is required when using this resin, and antibody function is preserved as IgGs are immobilized via the Fc region, keeping both antigen binding sites available for capturing targets.

CarboxyLink™ can be used to immobilize any protein or peptide via the C-terminus or aspartic or glutamic acid residues. Diaminodiproylamine (DADPA) is immobilized onto an agarose support. The carbodiimide crosslinker, EDC facilitates the formation of an amide bond between the carbon of the carboxylic acid and the nitrogen on the terminal amine of DADPA. This support is compatible with 50% organic solvent to improve immobilization of hydrophobic peptides.

SulfoLink™ Coupling Resin is iodoacetyl-activated agarose that reacts with reduced thiols to form a stable thioether linkage. CarboxyLink™ Coupling Resin is an amine-derivatized agarose that utilizes the carbodiimide crosslinker, EDC to conjugate the carbon of carboxylic acid to the nitrogen of the immobilized primary amine (hydrazide) to form a permanent amide bond.

SulfoLink™ Coupling Resin or UltraLink™ Iodoacetyl Resin would be good choices. They utilize the same chemistry to react with the reduced thiol group.

Please view our Tech Tip for assembling and packing a column.

This varies with the stability of the immobilized protein and the type of elution buffer used. Columns can typically be reused at least 10 times without significant loss of activity. If the immobilized molecule is sensitive to low pH, consider using our Pierce™ Gentle Ag/Ab Elution Buffer (Cat. No. 21013, 21027 or 21030). For alternative elution buffers, please refer to this Tech Tip.

Immobilized protein samples can be quantified using Pierce™ BCA Protein Assay or Coomassie Plus Protein Assay. Please refer to this Tech Tip on how to "Quantitate Immobilized Protein". Alternatively, this amount can be estimated by measuring the protein concentration in solution before and after coupling. The difference in the two measurements is the amount that is bound to the support

Our affinity supports are compatible with 3–4 M fresh guanidine or urea (Please note, aged solutions contain ammonia and will interfere with many binding reactions. Make up the guanidine or urea solution fresh prior to use). Alternatively, dissolve the peptide in 100% DMSO. Then add the peptide solution to coupling buffer so the DMSO concentration is 10– 30%.

UltraLink™ coupling resin is a hydrophilic, polyacrylamide/azlactone copolymer resin that has a high binding capacity with low nonspecific binding. It is a rigid support that is able to withstand high pressures (100 psi) and flow rates (3,000 cm/hr). This makes it ideal for the large-scale purifications often required by industrial customers. We offer UltraLink™ support versions of the typical beaded agarose AminoLink™, SulfoLink™ and CarboLink™ supports.

Coupling at pH 10 generally gives some increase in efficiency but may damage some ligands and requires a more involved procedure. If stability of the ligand is in question or the increase in coupling efficiency is of minor importance, use the pH 7.2 coupling procedure. This procedure has fewer steps and will still give good coupling efficiency.

No, the gel is stored in sodium azide with no loss in activity. Azide is not a primary amine and is rather unreactive.

No. Monoclonal antibodies are typically under-glycosylated, and coupling efficiency to CarboLink™ Gel may be low. However, many monoclonal antibodies can be immobilized using GlycoLink™ Gel. Try the Pierce™ Glycoprotein Carbohydrate Estimation Kit (Cat. No. 23260) to determine if your monoclonal antibody is glycosylated.

Yes, if too much sodium meta-periodate is added or if the incubation is performed too long, the oxidation begins to affect the amines on the protein and may cause the protein to break down or favor an amine linkage on to the resin. Furthermore, it may oxidize methionine residues if used in excess. We recommend a 10 mM solution of sodium meta- periodate for 30 minutes to control the reaction, oxidizing just the carbohydrate.

Yes, Chicken IgY can be immobilized using GlycoLink™ Gel, due to the high level of glycosylation of the IgY.

GlycoLink™ gel is hydrazide-activated crosslinked beaded agarose, and it is useful for coupling glycoproteins via aldehydes formed from their sugars by sodium meta-periodate oxidation. Reaction of aldehydes with hydrazide- activated resin is catalyzed by aniline resulting in >90% coupling in 4 hours or less. CarboxyLink™ gel on the other hand is crosslinked beaded agarose activated with diaminodipropylamine (DADPA) and is useful for immobilizing carboxyl- containing biomolecules after EDC activation. Actually, both resins can be used with EDC to couple ligands via carboxylic acids.

Note: Both immobilization chemistries are available on UltraLink™ Support as UltraLink™ Hydrazide and UltraLink™ DADPA respectively.

One milligram of peptide is sufficient for creating an effective affinity resin for purifying antibodies. Loading more peptide generally does not result in a significant increase of yield. Less than 1 mg of peptide can be used to prepare the column, but the yield of antibody will most likely decrease.

Please view our Tech Tip to prepare a reversible and measurable sulfhydryl-reactive affinity column.

Affinity chromatography uses a ligand that is coupled to a solid support. When a complex mixture is passed over the column, only those molecules having specific binding affinity to the ligand are bound and purified.

Affinity chromatography can be based on either positive selection or negative selection. This depends on what is bound to the column and the recovery method for the target of interest.

In Positive Selection, a ligand that will be the specific molecule(s) of interest is immobilized as bait to pull out the target molecule(s). Following the binding step, the non-bound molecules in the mixture are washed away, and the target protein is recovered using an elution buffer that releases the target from the bait. Elution can be performed by any means that will cause the bait molecule to release the target molecule. Common elution buffers are those that result in a change in pH, salt content, or otherwise result in a change of the three-dimensional structure of the bait and/or target, resulting in its release. Displacement by a small molecule that is structurally similar, such as glutathione for glutathione- s-transferase, is also a common elution method. Such displacement methods of elution are often more gentle and can result in a more functional target molecule upon recovery of the target from the affinity support. The majority of affinity chromatography is based on positive selection. To learn more about typical elution options for affinity purification, please refer to this Tech Tip.

In Negative Selection, a ligand is immobilized in order to bind and remove contaminating component(s) in the sample. Therefore, you eliminate what you do not want in your sample in order to keep what you need in a usable form. The use of negative selection protects the molecule of interest from exposure to elution conditions preventing possible denaturation or other damage, and reduction or elimination of downstream clean-up steps.

For general affinity purification or affinity chromatography of proteins, certain affinity pairs are recognized to work well for purification of a target molecule.

Common protein ligands include:

• Charged metal ions reactive with specific amino acids (e.g., Ni++/His purification)
• Other proteins with specific affinity to a natural or artificial feature of the target protein (streptavidin/biotin)
• Antibodies (immunoprecipitation)
• Fusion tags; small amino acids added to a target to allow for ease of purification. Fusion tags are often designed so that they can be readily removed from the target following purification.

• For purification of antibodies, we offer Melon™ Gel, which binds other proteins from serum, ascites, or culture supernatant, allowing the IgG to be collected in the flow-through. As low pH is typically used to elute IgG from Protein A, G, A/G or L, the use of Melon™ Gel can spare the IgG from exposure to such harsh conditions, which can be particularly important for monoclonal antibodies.

• For clean-up of samples prior to mass spectrometry analysis, we offer:

- Kits that remove highly expressed serum proteins such as the top 2 most abundant proteins (IgG and albumin) or top 12 abundant proteins

- Pierce™ Detergent Removal Spin Columns and Plates, and HiPPR™ Detergent Removal Spin Columns and Plates for removal of detergent

• Finally, our endotoxin removal resins (Pierce™ High Capacity Endotoxin Removal and Detoxi-Gel™ Endotoxin Removing Gel) allow clean-up of samples, which can be particularly important for proteins expressed in certain bacterial cell lines.

Batch method purification can be performed at any scale, however it is most commonly reserved for microcentrifuge tube–scale purifications involving 10–200 µL of resin. In batch method purification, wash and elution fractions are separated from the resin after centrifuging to pellet the resin beads. The liquid cannot be removed completely because some of it is contained within the volume of porous bead pellet. Consequently, a portion of each fraction about equal to the volume of resin used is left behind in the pellet, making washes and elution somewhat inefficient.

The spin cup purification method provides improved efficiency of wash and elution steps relative to the batch method. Centrifugation separates the liquid fraction by withdrawing it thoroughly from the resin, which is retained within the spin cup apparatus. Spin cup purification is most appropriate when 50–300 μL of immobilized ligand resin is used.

Read more about these purification methods in this Tech Tip.

An enterokinase cleavage site behind the DYKDDDDK (“FLAG”) tag can allow complete removal of the DYKDDDDK (“FLAG”) tag leaving no additional amino acids. We offer EKMax Enterokinase (Cat. Nos. E18001 and E18002) that can be used for this purpose. Subsequently, the EKMax Enterokinase can be removed using EK-Away Resin (Cat. No. R18001), a resin conjugated with soybean trypsin inhibitor, which has high affinity for enterokinase.

Yes. The UltraLink-based Anti-DYKDDDDH (“FLAG”) Affinity Resin (Cat. Nos. A36801, A36803, A36804) can be used for FPLC purification.

Here is the binding capacity: ≥3.2 mg DYKDDDDK-tGFP-His protein (˜32kDa)/mL settled beads.

Here is the binding capacity: ≥3.0 mg DYKDDDDK-tGFP-His protein (˜32 kDa)/mL settled resin.

Anti-DYKDDDDK Magnetic Agarose (Cat. Nos. A36797, A36798, A36799B) is offered as a 25% suspension (1 mL of 25% suspension = 0.25 mL of settled beads). UltraLink-based Anti-DYKDDDDK Affinity Resin (Cat. Nos, A36801, A36803, A36804) is offered as a 50% slurry (1 mL of 50% slurry = 0.5 mL of settled resin).

The High Select HSA/Immunoglobulin Depletion Resin is offered in 3 formats:

a) Resin alone: High Select HSA/Immunoglobulin Depletion Resin (Cat. No. A36368)
b) High Select HSA/Immunoglobulin Depletion Mini Spin Columns (Cat. Nos. A36365, A36366) that can process a maximum of 10 µL of human serum or plasma
c) High Select HSA/Immunoglobulin Depletion Midi Spin Columns (Cat. No. A36367) that can process a maximum of 100 µL of human serum or plasma. For serum samples containing abnormally high amounts of albumin or IgG, the sample load may need to be reduced.

No, we do not recommend reusing the High Select HSA/Immunoglobulin Depletion Resin.

No, the High Select HSA/Immunoglobulin Depletion Resin cannot be used for mouse samples; it is designed for use with human serum or plasma samples only.

The High Select Top 14 Abundant Protein Depletion Resin removes the following 14 proteins from human serum and plasma: human serum albumin (HSA), albumin, IgG, IgA, IgM, IgD, IgE, kappa and lambda light chains, alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-2-macroglobulin, apolipoprotein A1, fibrinogen, haptoglobin, and transferrin.

The High Select Top 14 Abundant Protein Depletion Resin is offered in 3 formats:

a) Resin alone: High Select Top 14 Abundant Protein Depletion Resin (Cat. No. A36372)
b) High Select Top 14 Abundant Protein Depletion Mini Spin Columns (Cat. Nos. A36369, A36370) that can process a maximum of 10 µL of human serum or plasma
c) High Select Top 14 Abundant Protein Depletion Midi Spin Columns (Cat. No. A36371) that can process a maximum of 100 µL of human serum or plasma. For serum samples containing abnormally high amounts of albumin or IgG, the sample load may need to be reduced.

Immobilized avidin, streptavidin or Neutravidin™ is used to purify biotinlyated molecules with high stringency. The high affinity of these molecules for biotin requires non-reversible denaturation to release biotin itself (including boiling in SDS-PAGE sample buffer or 8M guanidine, pH 1.5).

Please view our selection table to choose the right biotin-binding resin for your experiment.

Monomeric avidin is immobilized avidin denatured into monomers. It can be regenerated and reused, typically up to 10 purifications, and has a lower affinity compared to tetrameric biotin-binding proteins, allowing for much gentler recovery of biotinylated molecules.

The binding capacity of streptavidin agarose is 15–20 µg (18–24 nmoles) of fluorescein biotin per mL of sedimented gel.

Please refer to Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support for information.

pcDNA™ vectors with “capTEV™” tags (BioEase™ in vivo biotinylation peptide + 2 TEV protease recognition sites + 6X His tag) allow expression of biotinylated recombinant fusion proteins in mammalian cells. The protein complexes are purified under native conditions using streptavidin agarose included in the kit. 

The NativePure™ Columns hold up to 2 mL of chromatography resin and contain a 10 mL reservoir for sample or buffer. The column specifications are listed below: 

• Pore size of purification columns: 30–35 microns 
• Recommended flow rate: 0.5 mL/min 
• Maximum flow rate: 2 mL/min 
• Maximum linear flow rate: 700 cm/h 
• Column material: polypropylene 
• pH stability (long term): pH 3–13 
• pH stability (short term): pH 2–14 

CaptureSelect™ affinity resins can make the purification of biomolecules that do not have a traditional affinity purification solution much more efficient. When a targeted, specific affinity purification solution does not exist for a biomolecule, the protein purification scheme can be very complex needing 4–5 chromatography steps. Many chromatographic steps need to be utilized to separate the product of interest from key process and product related impurities. As the number of required purification unit operations increases, the product yield decreases and is heavily impacted with each step added. Yield drives cost of goods, so not having an affinity purification solution can greatly impact the cost for biotherapeutic manufacturing. Using in-house capabilities and expertise, CaptureSelect™ Custom Services design product-specific affinity ligand based on single-chain, camelid-derived antibodies, and couple that ligand to a high-performance, cost-effective affinity resin. We currently offer off-the-shelf bioprocess resins for antibody fragments, biosimilars, and viral vectors. Custom ligands can be used for biomolecule purification, scavenging of challenging impurities, and quantitation or detection of biomolecules. Custom resins are suitable for use in a cGMP production process.

Increased titers and product demand have caused substantial bottlenecks in downstream processing for a range of biomolecules. POROS™ chromatography resins address these challenges with solutions that help maintain performance and add process flexibility. Due to the polymeric nature of the backbone and the way we attach the ligands, the beads have very robust physical and chemical stability. This allows for aggressive solutions to be used, if needed, to improve cleanability and thereby increase resin lifetime/reuse. Our ion exchange resins are the go-to resins in downstream monoclonal antibody and recombinant protein chromatography where capacity, resolution, and yield are critical. We are also working on a new line of hydrophobic interaction resins that may be of interest for future projects that will be a best-in-class product for capacity and resolution with an added benefit of being able to load under lower salt concentrations and faster flow rates. We are also a strong partner when it comes to quality and supply of our products.

POROS™ resins are mechanically rigid and incompressible and can be packed effectively in low-pressure glass columns and in high-pressure stainless steel columns. The lack of wall support with increasing column diameter has minimal impact on chromatography performance because the beads support themselves, allowing for flexible column packing approaches and consistent and robust results. Columns can be packed with traditional flow pack, axial compression, or pack-in-place/stall pack packing  methods.

Resin lifetime depends on how the resin is used and the cleaning process that is employed. Therefore, each purification process will need to be evaluated specifically, especially if long lifetime is desired. Because binding can be different between resins, different cleaning schemes may be needed. An unoptimized cleaning process of any resin may yield <5 uses, and an optimized cleaning process can yield a hundred cycles or more. Discoloration of resin can occur for different reasons from process compounds such as metals like iron (Fe2+/3+) and copper (Cu2+), additives to the media (like vitamin B12 and folic acid), and elution solutions (like magnesium chloride, Mg2+). We can provide recommendations that have proved helpful for many customers to optimize the life of their POROS™ or CaptureSelect™ column.

In general POROS™ resins have behaved better for large biomolecules such as viral particles, fusion proteins, and globular proteins, where shape (not size) matters due to its unique pore structure and large pore structure as compared to traditional resins. As the target molecule size increases, capacities obtained will decrease. The large pore structure of POROS™ resins, which allows for convective flow (and therefore enhanced diffusion) is especially well suited for the purification of large biomolecules. POROS™ resins offer the best blend of high capacity (associated with chromatography beads) and improved chromatography efficiency (typically associated with monoliths or membranes). The average pore size for POROS™ resins is 1,000–3,600 Angstrom (100–360 nm) depending on the base bead/chemistry being utilized.

For example, the molecular weight of IgG is 150 kDa and the molecular radius is 55 Angstrom. The molecular weight of IgM is ~900 kDa (pentamer), and the molecular radius is 120 Angstrom. So both of these biomolecules can interact with the pore structure associated with POROS™ materials.

We recommend using 10 or 20 micron bead size for analytical work.

50 micron (or 20 micron for semi-preparative work) is better suited for scale-up due to the flow properties of the bead.

POROS™ resins rehydrate quite well. Here is the procedure that we recommend:

1. Flow 5% ethanol over the column at ≤350 cm/hour for 3 CVs, followed by water or 0.1 M sodium chloride at 300 cm/hour for 3 CVs.
2. If baseline noise is still present, flow 20% ethanol over the column at 300 cm/hour for 3 CVs, followed by water or 0.1 M sodium chloride at 300 cm/hour for 3 CVs.
3. If you have established column qualification tests and specifications, perform the tests after rewetting.

The physical stability of POROS™ resins is high. Customers have agitated, with overhead mixing and or bottom gassing for long periods of time (5–10 hours) and have performed hundreds of diaphragm pump passes during stall packs. Keep in mind that agitation with any device that creates grinding and sheer, such as with a magnetic stir bar, is not recommended for any type of resin.

Protein A and G will bind different types of antibodies differently depending on the species and class. For examples, Protein G will not bind IgM, IgA, or IgE antibodies, whereas Protein A will not bind IgG3 or Mouse IgG1, or any form of sheep, horse, or rat antibodies very well. Since most IgGs will bind to both Protein A and G, Protein A is usually chosen because it is less expensive than Protein G and can withstand harsher conditions for cleaning and regeneration, which is beneficial for biotherapeutic processes. Protein A also does not bind the antibodies as tightly as Protein G, making elution easier and allowing for higher recoveries and less drastic elution conditions.

UPLC is typically described as chromatography with sub-2 µm particles, which drives higher efficiency/better separation independent of operating flow rate. As particle size decreases, backpressure increases so the UPLC systems tend to have higher pressure ratings compared to HPLC systems. The flow rates on UPLC systems would be sufficient for POROS™ columns. Pump capabilities are typically 0.1–1.0 mL/min, so not as fast as a HPLC, but sufficient. Hold-up volumes on UPLC systems are lower than HPLCs, so this can help with sensitivity and efficiency. So from a pressure and flow standpoint there should be no challenge to operate a POROS™ CaptureSelect™ column on a UPLC system.

That being said, care should be taken running POROS™ columns on UPLC systems. Higher pressure safety interlock MUST be set at the rated column pressure, typically 180 bar. HPLC column hardware is not rated to operate at the static pressures possible and typical on UPLC systems. A clog or the like on a UPLC system could generate unsafe pressures on any HPLC-rated column before the default UPLC safety pressure interlock is tripped.

All of the pre-packed columns, regardless column size or resin, have the same 10–32 female fittings on the column. They are all compatible with the AKTA instrument platform, however due to the column volume of the 2.1 mmD x 30 mmL columns (0.1 mL), these will not perform well in AKTA or Avant purification systems due to the relative size of the hold- up volume in these systems. The smallest column (PEEK or SS) that could feasibly be used in these systems is the 4.6mmD x 50mmL (0.8 mL) format.

All of the pre-packed POROS™ columns, regardless of column size or resin, have the same 10–32 female fittings on the column. Columns are compatible with Waters™ and Agilent™ HPLC systems. You will need 1/16 inch male fittings for the HPLC to attach the column.

Protein A, Protein G, and Protein A/G bind almost exclusively to the IgG class of antibodies, but their binding properties differ among species and subclasses of IgG. Protein L binds in the variable fragment of some kappa light chains and can react with any immunoglobulin, not just IgG, as long as the correct kappa light chains are present. Protein L does not bind lambda light chains and certain kappa chains of different species.

• Protein A is generally preferred for rabbit, pig, dog, and cat IgG.
• Protein G has better binding capacity for a broader range of mouse and human IgG subclasses (e.g., IgG1 vs. IgG2)
• Protein A/G is a recombinant fusion protein that includes the IgG-binding domains of both Protein A and Protein
• G. Therefore, Protein A/G is ideal for binding the broadest range of IgG subclasses from rabbit, mouse, human, and other mammalian samples.
• Protein L binds to certain immunoglobulin kappa light chains. Because kappa light chains occur in members of all classes of immunoglobulin (i.e., IgG, IgM, IgA, IgE and IgD), Protein L can purify these different classes of antibody. However, only those antibodies within each class that possess the appropriate kappa light chains will bind. Generally, empirical testing is required to determine if Protein L is effective for purifying a particular antibody. It binds only Vk1 in mouse and VkI, VkIII and VkIV in human.

Read more about the general characteristics of Ig-binding proteins.

Protein A, Protein G, Protein A/G, and Protein L are all offered separately as purified protein, antibody purification kits, and covalently attached to agarose and other resins, magnetic beads and/or coated microplates. View the differentproduct categories and product detail information. Additionally, please refer to Protein Immunoprecipitation (IP), Co- Immunoprecipitation (Co-IP), and Puldown Support for information pertaining to our Dynabeads™ magnetic bead-based kits.

The Orientation kits allow for binding of an antibody to the Protein A or G resin. After the antibody is bound, an amine- reactive crosslinker (DSS) is added to covalently attach the antibody to the support. This allows for the target antigen to elute free of antibody contamination. The Protein A and G Orientation Kits are used for larger-scale purification of IgG than is typical in an immunoprecipitation.

The Pierce™ Antibody Clean-up Kit (Cat. No. 44600) was specifically designed for removal of amine containing molecules from IgG. Please view this Tech Tip for additional strategies on how to remove BSA and gelatin from antibody solutions.

For purification of antibodies, we offer Melon™ Gel, which binds other proteins from serum, ascites or culture supernatant, allowing the IgG to be collected in the flow-through. As low pH is typically used to elute IgG from Protein A, G, A/G or L, the use of Melon™ Gel can spare the IgG from exposure to such harsh conditions, which can be particularly important for monoclonal antibodies.

We offer a variety of resins targeting His-tagged fusion proteins or GST fusion proteins. Please see this selection table to choose the right resin for your experiments.

We also offer resins (both agarose and magnetic bead–based) with immobilized anti-c-Myc and anti-HA antibodies for targeting c-Myc and HA-tagged proteins respectively. Please check out the following links for the specific products:

• Anti-c-Myc agarose
• Anti-c Myc magnetic beads
• Anti-HA agarose
• Anti-HA magnetic beads:

Please see table below with the corresponding elution methods:

Agarose is generally used for smaller-scale purification at moderate flow rates, while superflow resin is optimized for larger-scale purification at high flow rates. Common table top microcentrifuges are often used for separation of the solid phase when using agarose resin, while the highly crosslinked form of the superflow resin imparts improved rigidity, enabling it to withstand high pressure and flow rates without compressing. This makes it easy to scale up from laboratory to industrial-scale purifications. Please read this brochure to learn more about the different resins.

Nickel has a higher affinity for histidines than does cobalt. It binds multiple histidines more tightly than cobalt, and requires more stringent conditions than is necessary for cobalt. The lower affinity of cobalt for multiple histidines typically results in less nonspecific binding of histidine-rich proteins that lack a his-tag compared to nickel resins.

HisPur™ Ni-NTA resin has a high capacity of up to 60 mg of 6xHis-tagged protein per milliliter. It is a versatile resin that can work under both native and denaturing conditions, and can also be used with a variety of lysis reagents and buffer additives.

HisPur™ Cobalt resin utilizes proprietary tetradentate chelating resin charged with cobalt. This system recovers highly purified protein with lower imidazole concentrations and has low metal leeching properties.

Both systems are qualified by purifying 2 mg of myoglobin protein on a column and performing a Bradford assay. Protein recovery must be 75% or higher.

The binding capacity is approximately 1–5 mg/mL.

Yes, empty plasmid columns (Cat. No. 1954124, 1954126) and ProBond™ resin (Cat. No. R80101) are available separately.

The ProBond™ resin is coupled with iminodiacetic acid groups (IDA). IDA is loaded with Ni2+ ions and binds Ni2+ by three coordination sites. The resin is used to purify 6x His-tagged overexpressed proteins. One mL should bind at least 1 mg of recombinant protein.

We have purified proteins with a minimum of 6 histidines. Some literature has indicated purification from as little as 4 or 5 histidines.

Hybrid purification is when lysis and binding are performed with the respective denaturing buffers, but the washing and elution step, however, are continued with the respective native buffer. You can also do the switch during the washing; do denaturing washing steps, switch to one or two native washing steps and elute with the native elution buffer.

Hybrid purification should be used when the protein of interest is insoluble, but the refolding is necessary to yield a functional protein for the activity assay. Instead of the hybrid method, you could also purify the protein under entirely denaturing conditions and refold later. This is recommended if high yields are required.

Please read our suggestions below:

• Maintain low protein concentrations 10–50 µg/mL.
• Disulfide bonds help to stabilize native proteins; add a redox pair such as GSH (reduced glutathione) and
• GSSG (oxidized glutathione) at a ratio of 10:1 with GSH concentration of 2–5 mM. Such a redox pair is helpful to create an oxidizing potential to break and build new S-S bonds during the folding process.
• Remove denaturants slowly by dilution or dialysis; glycine (50 mM, pH 9.0, 5 mM EDTA) helps to solubilize the proteins. If GuHCl (guanidinium HCl) is used, add 2 M urea because urea helps to stabilize the protein folding, too.
• Add detergents at a very low concentration such as 0.1–0.5% NP-40 or 0.005% (v/v) Tween™ 20.
• Include co-solvents to stabilize the proteins such as glycerol (5–20%) or PEG 8000 or glucose or sucrose (10 %)
• Certain anions (e.g., phosphate or sulfate) or cations (e.g., MES or HEPES) have positive effects, too; include salt and maintain a neutral pH such as 100 mM KCl, or 150–500 mM NaCl, 2 mM MgCl2
• Avoid protein degradation by adding protease inhibitors such as 0.5 mM PMSF, 0.005–2 µg/mL aprotinin, 2 µg/mL Pepstatin, or 2–5 µg/mL leupeptin.
• Dialysis of a phosphate buffer when against calcium will result in a calcium phosphate precipitate. If CaCl2 is needed for subsequent enterokinase digestion (10 mM Tris pH 8, 10 mM CaCl2), remember to add CaCl2 after dialysis is complete.

We offer a Quant-iT™ Protein Assay Kit (Cat. No. Q33210), which is more sensitive than standard absorbance-based assays and can quantitate proteins from 0.25–5 µg. The signal is unaffected by many common contaminants, such as DTT, beta-mercaptoethanol, amino acids, and DNA. Imidazole at a final concentration below 1.25 mM is acceptable. Above that concentration, the imidazole begins to interfere with the assay.

Please note, imidazole does absorb at 280 nm, and the absorbance varies with concentration. So to be perfectly accurate, each eluted fraction should be blanked against its elution buffer. The imidazole absorbance in 20 mM sodium phosphate, 500 mM NaCl, pH 6.0 is shown below:

ProBond™ resin can be used for up to three or four purifications of the same protein without recharging. We only recommend reusing the resin for purification of the same recombinant protein. If you want to reuse the resin, wash it with 0.5 M NaOH for 30 minutes and equilibrate the resin with the appropriate binding buffer.

Please keep in mind, while the ProBond™ resin is rechargeable, the Sepharose™ medium will sustain wear. If the resin turns white due to the loss of nickel ions from the column, recharge the resin.

To recharge 2 mL of resin in a purification column:

1. Wash the column two times with 8 mL 50 mM EDTA to strip away the chelated nickel ions.
2. Wash the column two times with 8 mL 0.5 M NaOH.
3. Wash the column two times with 8 mL of sterile, distilled water.
4. Recharge the column with two washes of 8 mL NiCl2 hexahydrate at a concentration of 5 mg/mL prepared in sterile, distilled water.
5. Wash the column two times with 8 mL distilled water.
6. Add 0.02% azide or 20% ethanol as a preservative and cap or apply a Parafilm™ cover to the column. Store at room temperature.

The columns are 9 cm high, conical 0.8 x 4 cm of polypropylene and hold up to 2 mL of resin and 10 mL of eluent or sample. The average pore size of the column filter is 30–35 microns.

Yes, the maximum flow rate is 4 mL/hr (1 mL column) and the maximum linear flow rate is 700 cm/hr in an XK16/60 column with a 5 cm bed height. We have successfully used a flow rate of 2 mL/min. The maximum pressure is 0.3 MPa (3 bar, 42 psi). Do not run above 50–100 psi. The pH stability for long term is 3–13 and 2–14 for short term.

EDTA and EGTA can be used at up to 1 mM concentration. Higher concentrations will strip the resin (i.e., the nickel ion will leach out).

Up to 20 mM beta-mercaptoethanol can be used. DTE (dithioerythritol) or DTT (dithiothreitol) can also be used up to a concentration of 1 mM.

Non-ionic detergents including Triton™, Tween™, NP-40™ detergents can be used at up to 2% concentration to remove background proteins and nucleic acids. Cationic detergents can be used at up to 1% concentration. Anionic detergents such as SDS or Sarkosyl™ detergent are not recommended. Zwitterions (such as CHAPS) can be used at up to 1% concentration.

Please see the suggested reagents below:

• Up to 60 mM citrate has been used successfully
• Up to 20% ethanol prevents hydrophobic interaction between proteins
• Up to 50% glycerol prevents hydrophobic interaction between proteins
• Imidazole can be used at low concentrations (20 mM) to inhibit nonspecific binding and at higher concentrations (>100 mM) to elute the 6xHis-tagged proteins.
• Up to 2 M sodium chloride prevents ionic interactions; you should use at least 150 mM

Up to 6 M guanidinium chloride or up to 8 M urea can be used.

This can be achieved, though not effectively. DMEM and other mammalian tissue culture media contain many contaminating proteins (and free amino acids like glutamine and histidine) that may compete for binding to the ProBond™ resin. For best results, mammalian tissue culture media should be dialyzed (against binding buffer) or filtered prior to loading on equilibrated ProBond™ resin. Other methods such as ion exchange or sizing columns can help to prepare the protein for effective isolation on the ProBond™ column. If necessary, the culture media may be adjusted to facilitate direct binding to the column. The pH of the media should be adjusted to 7.5–8.0. The salt concentration in the media should be 0.5 M NaCl.

Purification may be performed at 4 degrees C or room-temperature depending upon the sensitivity of the synthesized product.

1. Upon completion of incubation, remove the desired portion of reaction for His-tag purification to a clean microcentrifuge tube. Add 4 volumes of Binding buffer and vortex briefly (Add 200 μL for 50 μL of reaction). Centrifuge 5 minutes at 12,000 rpm.
2. Transfer the supernatant to a 2.0 mL tube containing 50 μL pre-equilibrated resin.
3. Incubate with shaking or mixing for 30–60 minutes.
4. Spin down resin for 2 minutes at 800 x g. Do not spin any higher or the resin will collapse and recovery will be low. Carefully  remove supernatant.
5. Add 200 μL wash buffer and mix for 5 minutes.
6. Spin down resin for 2 minutes at 800 x g. Carefully remove supernatant.
7. Repeat steps 5 and 6.
8. Add 100 μL Elution Buffer and mix for 5 minutes.
9. Spin down resin for 2 minutes at 800 x g. Carefully remove and save supernatant.
10. Repeat steps 8 and 9.

Binding Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
6 M guanidine HCl (optional)**

Wash Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
15–25 mM imidazole*

Elution Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
150–250 mM imidazole*

**Depending on downstream applications, the purification may be performed under semi-denaturing conditions, or native conditions. Under semi-denaturing conditions, dilute the reaction in denaturing Binding Buffer containing 6 M guanidine HCl; then wash and elute with native buffers.

The concentration of imidazole is dependent upon the type of resin used. For Ni-NTA or ProBond™ resins, use 25 mM imidazole in the wash buffer and 250 mM imidazole in the elution buffer.

When purifying His-tagged proteins proteins from E. coli lysates, keep in mind that there is a 29 kDa endogenous protein SlyD. SlyD has a histidine-rich c-terminus and is found in all strains of E. coli and Salmonella. The contamination is apparent when the His-tagged protein is expressed at a low level or not expressed at all. In such cases, SlyD will bind to the nickel column with great affinity. Increase the purification stringency to overcome SlyD binding.

If protein is released into LB media from E. coli, try native isolation conditions. Dialyze against binding buffer and possibly concentrate before going on to the ProBond™ resin (10% glycerol in the dialysis binding buffer will concentrate the secreted protein well). Another option is to add about 24 g NaCl and 2.8 g Na2HPO4 per liter of media, and adjust the pH to 7.8 with NaOH or HCl. This will turn the media into pseudo-binding buffer (~500 mM NaCl, ~20 mM NaPO4, pH 7.8); perform binding, washing, and eluting with either imidazole or by altering pH.

A simple and reliable method for precipitating protein from bacterial culture supernatants based on a pyrogallol red- molybdate-methanol (PRMM) protocol has been developed and applied for the analysis of proteins secreted by a bacterial type III secretion system (Caldwell RB, Lattemann CT (2004). Simple and reliable method to precipitate proteins from bacterial culture supernatant Appl Env Micro 70(1):610–612).

Over-expressed S. cerevisiae proteins should be purified as described on Pages 50 and 51 in the Pichia manual, (see the section under Purification). However, if the breaking buffer is used for S. cerevisiae, the EDTA should be omitted. In other words, if working with Pichia, the breaking buffer should contain ETDA (see Page 59 of the Pichia manual for the Breaking Buffer recipe), but EDTA should be omitted if working with S. cerevisiae.

Alcohol oxidase (AOX protein) is an octamer and has at least a few His stretches. Hence, AOX protein will bind to the ProBond™ resin. In order to prevent co-elution, we recommend that you perform ion exchange purification prior to the ProBond™ purification. You will need to know the pI of the expressed protein for good binding and need to optimize the ion exchange step for efficient separation from AOX.

We recommend purifying His-tagged Pichia proteins using the protocol described on Pages 50 and 51 in the Pichiamanual , under Purification. It describes how to obtain the supernatant (soluble proteins) and pellet (urea/insoluble proteins) by using the breaking buffer (BB). The composition of the breaking buffer is listed on Page 59 of the Pichiamanual.

We offer the following products:

• AcTEV™ Protease (Cat. Nos. 12575015,12575023)
• EKMax™ Enterokinase (Cat. Nos. E18001, E18002)
• SUMO Protease (Cat. No. 12588018)

The recognition site for AcTEV™ Protease is ENLYFQS/G

EKMax™ enterokinase is a recombinant preparation of the catalytic subunit of bovine enterokinase. EKMax™ enterokinase recognizes the sequence DDDDK and cleaves the peptide bond after the lysine residue.

We offer EK-Away™ Resin that specifically binds EKMax™ Enterokinase, and can be used to remove it after cleavage.

SUMO protease, also known as Ulp, is a recombinant fragment of ULP1 (Ubl-specific protease 1) from Saccharomyces cerevisiae. It is highly specific for the SUMO protein fusion, recognizing the tertiary structure of SUMO rather than an amino acid sequence. The SUMO protease itself has a His tag for easy removal from the protein mix after cleavage.

We would recommend using our SUMO system.

Enrichment of specific proteins or protein complexes can most easily be accomplished by using target-specific immobilized antibody or PTM specific affinity binding. We offer several kits targeting phosphorylated proteins, alpha- linked mannose and terminal glucose residues, N-acetyl glucosamine, polyubiquinated proteins, ATP binding sites, and active serine hydrolase enzymes. Choose the right protein enrichment kit for your application.

The columns in the kit contain a proprietary metal that interacts with negative charges from phosphate groups. The optimized buffer conditions enable specific capture of phosphoproteins from complex biological samples.

From 2 mg of total protein, the expected yield is 300 µg when using the Pierce™ Phosphoprotein Enrichment kit.

The ConA kit (Cat. No. 89804), which has concanavalin A immobilized on the resin, recognizes α-linked mannose, and to a lesser extent, terminal glucose residues. The WGA kit (Cat. No. 89805), which has wheat germ agglutinin immobilized on the resin, recognizes N-acetyl glucosamine (GlcNAc) groups and sialic acid.

Thermo Scientific Pierce™ Active Site Probes are chemical probes that covalently bind to the active sites of specific enzyme classes such as kinases, GTPases, and serine hydrolases. These probes can be used to selectively enrich, identify, and profile target enzyme classes across samples or assess the specificity and affinity of enzyme inhibitors.

The structure of the probes consists of an active site specific reactive group, a linker region, and a tag group for detection or affinity capture. Active site reactive groups are typically electrophilic compounds that covalently link to nucleophilic residues in enzyme active sites. All active site probes can be used to determine inhibition of enzymes by small molecules, and some probes also preferentially react with only active enzymes, allowing for activity-based proteomic profiling (ABPP). Read more about active site probes.

Active site probe kits enable the following:

• Broad enrichment of nucleotide binding proteins, including kinases and G-proteins from tissues, cells, and subcellular proteomes
• Dose-dependent profiling of small molecules
• Enrichment of enzymes based on function
• Profiling of dozens to hundreds of inhibitor targets and off-targets

We offer standalone protease inhibitors (AEBSF, Aprotinin, PMSF, EDTA, etc.), as well as cocktails or combinations of protease inhibitors in liquid, tablet, and larger capsule formats. These cocktails of protease inhibitors can also be purchased in combination with a cocktail of phosphatase inhibitors, in both liquid and tablet form, for even greater convenience.

The Pierce protease, phosphatase, as well as (protease and phosphatase) inhibitors are offered as mini tablets, with or without EDTA. Each mini tablet is sufficient for 10 mL of solution and is suitable for reagent preparation prior to protein extraction for cultured cells and tissues. In addition, the Pierce protease inhibitor tablets are offered with or without EDTA and each tablet is sufficient for 50 mL of solution.

Here are the catalog numbers:
-Pierce Protease Inhibitor Mini Tablets (Cat. No. A32953)
-Pierce Protease inhibitor Mini Tablets, EDTA-free (Cat. No. A32955)
-Pierce Phosphatase Inhibitor Mini Tablets (Cat. No. A32957)
-Pierce Protease and Phosphatase Inhibitor Mini Tablets (Cat. No. A32959)
-Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free (Cat. No. A32961)
-Pierce Protease Inhibitor Tablets (Cat. No. A32963)
-Pierce Protease Inhibitor Tablets, EDTA-free (Cat. No. A32965)

Metalloproteases bind metal ions in their active sites. EDTA is a metalloprotease inhibitor that acts by chelating metal ions that are required by these proteases for activity. We recommend using the protease or phosphatase inhibitor tablet or mini tablet containing EDTA if any metalloprotease is likely to adversely affect the functioning of the target protein. However, it is important to avoid using EDTA when the downstream application involves metal chelation affinity purification or if the target protein requires metals ions for proper function.

The Pierce protease, phosphatase and (protease and phosphatase) inhibitor tablets and mini tablets are compatible with common protein assays such as BCA Protein Assay, 660 nm Protein Assay, Coomassie Plus (Bradford) Assay, and Detergent Compatible Bradford Assay.

The Pierce protease, phosphatase, as well as (protease and phosphatase) inhibitor tablets and mini tablets are stable for at least one year when stored at 4 degrees C, but we only recommend preparing fresh solutions just before needed. If you are interested in liquid protease or phosphatase inhibitors, please use our inhibitor cocktails.

The final concentration of each component in the protease and phosphatase inhibitor tablets and mini tablets is proprietary. We release only the identities of the inhibitors.

Yes, the following components of the Pierce protease, phosphatase and (protease and phosphatase) inhibitor tablets and mini tablets are offered separately:

- AEBSF (Cat. No. 78431)
- Aprotinin (Cat. No. 78432)
- Bestatin (Cat. No. 8433)
- E-64 (Cat. No. 78434)
- Leupeptin (Cat. No. 78435)
- Pepstatin A (Cat. No. 78436)
- EDTA (Cat. No. 17892).

Yes, our Pierce Protease Inhibitor XL Capsules, EDTA-Free (Cat. No. A37989) are designed for this purpose. Each capsule is sufficient for 500 mL of solution.

We do not recommend “splitting” the capsule into smaller portions. Researchers using less than 500 mL should choose a different format (liquid or tablet) of protease inhibitors.