5 Different Types of Chromatography Resins, How to Choose?

Chromatography technology plays a critical role in the biopharmaceutical sector, by offering an efficient approach to achieve the separation of complex components. Chromatography resin consists of ligands and a base matrix, which serves as a critical factor in the downstream processing by determining the productivity and quality of pharmaceutical ingredients purification. 

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Chromatography resins can be categorized into affinity resin, ion exchange (IEX) chromatography resin, hydrophobic interaction chromatography (HIC) resin, Size exclusion chromatography (SEC) resin, and Mix-mode resin in terms of separation mode.

From this article you will learn:

1. Overview of chromatography resins
2. Introduction to 5 common types of chromatography resins
3. How do you choose between different types of chromatography resins?
4. Outsource your resin needs to Bestchrom

Overview of chromatography resins

Chromatography resins are a class of key materials used in chromatography processes and are widely used in the pharmaceutical, biotechnology and chemical fields. These resins are often highly selective and can effectively separate compounds in mixtures.

The choice of chromatography resin depends on the desired separation properties, including molecular size, charge, hydrophilicity and hydrophobicity, etc. Common chromatography resin types include ion exchange resins, hydrophilic and hydrophobic chromatography resins, and metal chelate chromatography resins.

These resins play a key role in the preparation and purification of biomolecules, drugs and compounds, improving the efficiency and effectiveness of chromatography processes in laboratories and industry.

Introduction to 5 common types of chromatography resins

There are different types of chromatography resins, and each resin has its own advantages and functions according to user requirements.

Affinity chromatography

Affinity chromatography is a chromatography method separating biomolecules based on the specific interaction among biomolecules. Thanks to its high selectivity, affinity resin is able to capture protein from complexes at a purity higher than 90% via one-step purification. 

Affinity resin is widely applicable in the efficient purification of antibodies, tag proteins, and other bio-molecules with specific adsorption.

Advantages: High selectivity, excellent purity outcomes, and suitability for purifying specific target molecules.

Ion exchange (IEX) chromatography

Ion exchange (IEX) chromatography is a separation method based on the different mass and quantity of electric charges on biomolecules. Enjoying advantages including high selectivity, high binding capacity, high yield, and convenient operation, the versatile chromatography method can be used in the initial capture step as well as intermediate purification and polishing. 

Therefore, it is widely applicable in the purification of electric-charged bio-molecules including amino acids, peptides, antibodies, proteins, saccharides, viruses, and nucleotides.

Advantages: High specificity for charged molecules, effective separation of biomolecules, and applicability in both analytical and preparative chromatography.

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a widely used method in the separation of macro-biomolecules based on the hydrophobicity difference on the molecule surface.  The biggest merit lies in its mild interaction with proteins, which enables the maintenance of natural structure and bio-activity in macro-biomolecules. 

HIC adopts an adsorption mode in terms of high salinity sample loading and low salinity elution, which makes it an ideal purification option after eluting with high salinity. HIC provides an efficient method for the separation of macro-molecules including antibodies, recombinant proteins, vaccines, peptides, and nucleic acid.

Advantages: Gentle separation conditions, effective purification of proteins, and compatibility with proteins that denature in other chromatographic methods.

Size exclusion chromatography (SEC)

Size exclusion chromatography (SEC), also known as gel filtration, is a non-adsorption chromatography method. It achieves chromatography separation based on the size and shape of biomolecules, which means the key to SEC resin selection lies in the right fractionation range. 

SEC enjoys the advantage of easy operation, which enables the completion of chromatography separation via a single buffer. Therefore, it is an ideal option in the desalting, buffer exchange, and polishing step of chromatography.

Advantages: Non-destructive separation method, compatibility with a wide range of biomolecules, and suitability for determining molecular weight.

Mixed-mode chromatography

Mixed-mode chromatography is an innovative chromatography method that can simultaneously provide different interactions for the binding between ligands and macro-biomolecules. Dominant interactions in this category include electric charge interaction and hydrophobic interaction. 

Mixed-mode chromatography enjoys wider binding condition and a bigger operation room, which simplifies process step and boost productivity. It is widely applicable in the purification of viruses, antibodies, peptides, recombinant proteins, and nucleic acids, especially in providing effective solutions to challenges faced in the purification process.

Advantages: Versatility in separating complex samples, increased selectivity, and the ability to handle challenging separation tasks.

How do you choose between different types of chromatography resins?

1. Affinity Chromatography:

Use When: High specificity is required for the separation of target molecules based on specific binding interactions.

Ideal for: Purification of proteins, antibodies, and enzymes where a strong affinity between the target and ligand is present.

2. Ion Exchange (IEX) Chromatography:

Use When: Separation based on charge differences is needed, making it suitable for molecules with varying ionization states.

Ideal for: Purification of proteins, peptides, and nucleic acids by exploiting differences in charge.

3. Hydrophobic Interaction Chromatography:

Use When: Separation is desired based on hydrophobicity, offering a milder condition compared to other hydrophobic methods.

Ideal for: Purifying proteins that are sensitive to high salt concentrations or denaturation.

4. Size Exclusion Chromatography (SEC):

Use When: Separation based on molecular size is crucial, allowing for the analysis of size distributions and purification of biomolecules.

Ideal for: Determining molecular weights and separating biomolecules based on their size without denaturation.

5. Mixed-Mode Chromatography:

Use When: Enhanced selectivity is needed by combining different separation mechanisms like ion exchange, hydrophobic interaction, and affinity.

Ideal for: Complex samples requiring versatile approaches, providing increased selectivity in bioseparation processes.

Chromatography resin plays a critical role in chromatography purification. Different combinations of functional groups and base matrices provide various functions to resins, which enables the efficient purification of complex components via a selection of the right resin. 

When choosing suitable resins, the following factors should be taken into account:  matrix property (polymer-based matrix provides a high flow rate due to its good mechanical property; Agarose-based resin enjoys better non-specific adsorption due to its excellent hydrophilicity), bead size(finer beads provide high selectivity while big beads can endure high flow rate), resin binding capacity (which depends on ligand types and ligand concentration) as well as scalability. 

In addition, evaluation results in terms of sample yield and quality should also be considered when making the resin purchase decision.

NUPTEC supply professional and honest service.

Outsource your resin needs to Bestchrom

Bestchrom Biosciences focuses on the process development and product R&D required in bio-pharmaceutical downstream processing. We take our core strength in product quality and production capacity, which enable us to continuously provide chromatography resins with more variety, better performance, and cost-effectiveness. 

Contact us today by sending an or visit our resin product page!

References

Ion exchange resin

Affinity chromatography

Mixed-mode Resins

Size exclusion resins

Extract and stabilize your target protein from the sample

Protein extraction techniques vary depending on the source of the starting material, the location of the protein of interest within the cell, and the downstream application. Other important considerations include the preservation of protein activity and function as well as the reduction of background effects.

Protein extraction

Tissue and cell lysis

Historically, mechanical disruption has been used to lyse cells and tissues; our gentle, detergent-based solutions have been developed to efficiently lyse cells and enable the separation of subcellular structures without requiring physical disruption, providing high yields of active proteins.

Protein extraction product features:

• Optimized—formulations maximize protein yield and preserve protein activity
• Efficient—only produces minimal cross-contamination between subcellular fractions
• Compatible—extracts can be used directly in most downstream applications
• Gentle—eliminates the need for mechanical cell disruption for most sample types

High protein yield from a variety of mammalian cell types and cellular compartments

Protein extraction efficiency from major cellular compartments using M-PER Mammalian Protein Extraction Reagent. Lysates from established cell lines and primary cultures were prepared using M-PER reagent and extraction efficiency from the various cellular compartments evaluated. For each target protein, 10 µg of lysate was loaded for and electrophoresed by SDS PAGE, transferred to nitrocellose membrane and detected by western blot using SuperSignal West Pico PLUS Chemiluminescent Substrate.

Protein yield from various cell types using M-PER Mammalian Protein Extraction Reagent. Cells were harvested at 85% confluency, washed twice and collected in ice-cold PBS and counted. For each cell type, 1 x 106 cells were pelleted by centrifugation at 2,000 x g for 5 minutes and lysed in 1 mL M-PER Reagent for 5 minutes. The cell lysates were clarified by centrifugation at 14,000 x g for 10 minutes and the supernatant was collected and the protein concentration (µg/million cells) was determined using the Pierce BCA Protein Assay.

Efficient and selective enrichment of membrane proteins

Improved protein yield using the Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit. Membrane proteins were isolated from mouse liver tissue and HeLa cells using Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit and three other commercial extraction kits. Protein yields (μg) for membrane, cytosolic, and total fractions were determined using the Thermo Scientific Pierce BCA Protein Assay Kit.

Efficient protein extraction from a variety of tissue types

Tissue cell lysis protein yield with T-PER Tissue Protein Extraction Reagent. Duplicate tissue samples were weighed, resuspended in 1:10 to 1:20 w/v T-PER Reagent and disrupted in a chilled Dounce or benchtop tissue homogenizer. The resulting lysates were centrifuged at 10,000 x g for 5 minutes and the supernatant was collected. The protein concentration of each lysate was determined using the Pierce BCA Protein Assay to determine protein yield per milligram of starting tissue.

Efficient extraction from bacterial cells

Protein yield comparison of two bacterial cell lysis reagents.E. coli ER/pLATE51-Klenow, ER/pGST-CC-StpB, and ER/pGS-Syk cell pellets (0.5 g), were resuspended in 2.5 mL aliquots of Thermo Scientific B-PER Complete Bacterial Protein Extraction Reagent or EMD Chemicals BugBuster Master Mix with gentle vortexing for 15 minutes at room temperature. Insoluble cell debris was removed by centrifugation at 16,000 x g for 20 minutes at 4°C. Protein yields (concentrations) for soluble fractions were determined using the Pierce BCA Protein Assay Kit.

Sample typeGoalProduct highlightPrimary or cultured mammalian cells or tissuesTotal protein extractionM-PER reagent
T-PER reagent
N-PER reagentRIPA Lysis and Extraction Buffer
IP Lysis BufferCultured mammalian cells or tissuesSubcellular fractionation or organelle isolationNE-PER reagent
Subcellular Fractionation Kits
Mitochondria Isolation KitsGPCR Extraction and Stabilization Reagent
Cell Surface Protein Isolation Kit
Syn-PER Reagent
Lysosome Enrichment KitBacterial cellsTotal protein extractionB-PER reagentYeast cellsTotal protein extractionY-PER reagentInsect cells (baculovirus)Total protein extractionI-PER reagentPlant tissue (leaf, stem, root, flower)Total protein extractionPlan Total Protein Extraction Kit

Detergent solutions

Detergents are frequently used in cell lysis reagent formulation and other protein research methods. Thermo Scientific Surfact-Amps Detergent Solutions are highly purified, precisely diluted (10%) formulations that are ideal for applications or assays that are sensitive to contaminants present in unpurified detergents.

Protein detergent product features:

• Accurate—precise 10% detergent solution in ultrapure water
• Easy-to-use—solution is simple to dispense and dilute
• Exceptionally pure—less than 1.0 μq/mL peroxides and carbonyls
• Stable—packaged under inert nitrogen gas in glass ampules or HDPE bottles

Generic structure of a detergent molecule.

Protein stabilization

Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and function. To prevent these negative effects, protease and phosphatase inhibitors can be added to the lysis reagents. Numerous compounds have been identified that can inactivate or block the activities of proteases and phosphatases.

Protease and phosphatase inhibitor product features:

• Convenient—ready-to-use, fully disclosed, broad-spectrum formulations available as either liquid cocktails, tablets, or capsules, in multiple pack sizes and with a minimum of 1 year of shelf life
• Complete protection—all-in-one formulations containing both protease and phosphatase inhibitors are offered in both liquid and tablet formulations (with EDTA or EDTA-free)
• Compatible—use directly with Thermo Scientific Pierce Cell Lysis Buffers, other commercial, or homemade detergent-based lysis reagents

Broad effective inhibition of proteases and phosphatases

Performance comparison between three commercially available protease inhibitor tablets. Pancreatic extract (100 μL; 0.5 μg/μL) was incubated with quenched fluorescent protease-cleavable substrates for trypsin, cysteine, and metalloprotease and cathepsins, in the presence of the reformulated Thermo Scientific Pierce Protease Inhibitor Mini Tablets, Roche™ Complete™ Protease Inhibitor Tablets, and Sigma-Aldrich™ SIGMAFAST™ Protease Inhibitor Cocktail Tablets, with and without EDTA. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate emission wavelengths. The percent inhibition is shown for each protease inhibitor formulation.

Protein phosphorylation in cell extracts is broadly preserved by Thermo Scientific Phosphatase Inhibitor Mini Tablets.(A) HCT116 cells were serum-starved, then either treated with EGF for 15 min or left as control cells. Cell lysates were prepared in Thermo Scientific Pierce IP Lysis Buffer with Thermo Scientific Protease and Phosphatase Inhibitor Mini Tablets, EDTA-Free, or with no inhibitor. Lysate containing 500 μg of protein was then incubated with 5 μg of phospho-tyrosine antibody overnight at 4ºC. The complex was then incubated with Thermo Scientific Pierce Protein A/G Magnetic Beads for 1 hr at room temperature. Beads were washed, and low-pH elution was performed. The eluates were subjected to western blotting, and the membrane was then probed with EGFR antibody for chemiluminescence detection. (B) The degree of inhibition for protein, alkaline, and acid phosphatase activity was determined in kidney extract (25 μL; 0.5 μg/μL) by incubating extracts with a fluorogenic substrate (MFP or FDP) that measures phosphatase activity upon dephosphorylation in the presence of Pierce Phosphatase Inhibitor Mini Tablets, Roche™ PhosStop™ Phosphatase Inhibitor Tablets, and Sigma-Aldrich™ Phosphatase Inhibitor Cocktail 2 and 3 liquid formulations. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate emission wavelength. The percent inhibition is shown for each phosphatase inhibitor formulation.

Tips

• Cell lysis disrupts cells membranes and organelles resulting in unregulated proteolytic activity that can reduce protein yield and function. To prevent extracted protein degradation, it is often necessary to add protease and phosphatase inhibitors to cell lysis reagents.
• Most researchers use a mixture of several different inhibitor compounds to ensure the protein extracts do not degrade before analysis of the target of interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
• Analyze a sample of the solubilized protein and the insoluble fractions by SDS-PAGE to determine the efficiency of the protein extraction method used.
   

Protein extraction and stabilization products

Tissue and cell lysis

Product selection guidesProductsMammalian cell protein extractionT-PER Tissue Protein Extraction Reagent
N-PER Neuronal Protein Extraction Reagent
M-PER Mammalian Protein Extraction Reagent
RIPA Lysis Buffer
IP Lysis BufferInsect protein extractionI-PER Insect Cell Protein Extraction ReagentBacterial cell lysisB-PER Complete Bacterial Protein Extraction Reagent
B-PER Bacterial Protein Extraction Reagent
B-PER (PBS) Bacterial Protein Extraction Reagent
B-PER II (2X) Bacterial Protein Extraction ReagentPlant protein extractionPlant Total Protein Extraction KitYeast protein extractionY-PER Yeast Protein Extraction ReagentSubcellular fractionationNE-PER Nuclear and Cytoplasmic Extraction Reagents
Subcellular protein fractionation kits (tissue or cultured cells)
Syn-PER Synaptic Protein Isolation KitMembrane protein extraction and isolationGPCR Extraction and Stabilization Reagent
Mem-PER Plus Membrane Protein Extraction Kit
Cell Surface Protein Isolation KitOrganelle isolationLysosome Enrichment Kit for Tissues and Cultured Cells
Organelle isolation using magnetic beads
Mitochondrial isolation kits (tissue or cultured cells)Neuronal cell protein extractionN-PER Neuronal Protein Extraction Reagent
Syn-PER Synaptic Protein Isolation Kit

Detergents for protein solubilization

Product selection guidesProductsDetergents for protein solubilizationSurfact-Amps detergents
n-Dodecyl-beta-maltoside detergent
CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)
Sodium dodecyl sulfate (SDS)
Octylthioglucoside (OTG) detergent
Octyl-beta-glucoside detergent

Protease and phosphatase inhibitors

Product selection guidesProductsProtease and phosphatase inhibitorsProtease liquid cocktails, tablets, and capsulesHalt Protease Inhibitor Cocktail
Halt Protease Inhibitor Cocktail, EDTA free
Pierce Protease Inhibitor tablet
Pierce Protease Inhibitor mini tablet
Pierce Protease Inhibitor tablets, EDTA-free
Pierce Protease Inhibitor mini tablets, EDTA-free
Pierce Protease Inhibitor XL Capsules, EDTA-freePhosphatase liquid cocktail and tabletsHalt Phosphatase Inhibitor Cocktail
Pierce Phosphatase Inhibitor TabletCombined protease and phosphatase liquid cocktails and tabletsHalt Protease and Phosphatase Inhibitor Cocktail
Halt Protease and Phosphatase Inhibitor Cocktail, EDTA free
Pierce Protease and Phosphatase Inhibitor Mini Tablet
Pierce Protease and Phosphatase Inhibitor Mini Tablet, EDTA Free

Purify and enrich your protein sample

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. Ion exchange and affinity chromatography are two commonly used strategies for partial or 1-step purification.

Affinity purification

Also known as affinity chromatography, this purification method is enabled by the specific binding properties of a protein to an immobilized ligand. Since the protein of interest is tightly bound, contaminants can be removed through wash steps, and the bound protein can be stripped (eluted) from the support in a highly purified form. Affinity purification is desirable because it often produces higher protein yields and requires less steps than other purification methods. It is the method of choice for purifying recombinant or biotinylated proteins and antibodies.

Discover more about protein purification
See protein isolation and purification learning resource

Ion exchange (IEX) purification

Also known as ion exchange chromatography, this purification method enables the separation of proteins based on the protein charge at a particular pH. Since multiple proteins may have similar charges, IEX chromatography generally enables only partial purification of a protein of interest when used early in a multistep purification process. However, IEX resins can also be used during a final polishing step to remove specific contaminants that persist after other purification steps. Typically, proteins bind to the IEX column at low ionic strength and elute differentially by increasing salt concentration or changing pH in a gradient. A cation exchange resin binds to positively charged proteins; an anion exchange resin binds to negatively charged proteins. Ion exchange resins are classified as “weak” or “strong”, which refers to the extent that the ionization state of the functional groups varies with pH.

Discover more about ion exchange purification

How protein purification works

1. Bind: Capturing protein of interest using a specific ligand.
2. Wash: Remove unwanted proteins and other impurities, such as nucleic acids, during the wash phase.
3. Elute: This step reverses the binding reaction. One can elute and enrich the target protein by varying buffering conditions such as pH, salt concentration, and detergents, or including competitive binders.
4. Polishing: Using an additional chromatography step (usually ion-exchange or size exclusion) to remove remaining trace impurities and other contaminants by making best use of the known characteristics of the target protein.

Protein purification workflow

Comparison of DYKDDDDK-tagged SUMO protein yield and background using Pierce anti-DYKDDDDK resin and other products C- and N-terminal DYKDDDDK-tagged SUMO proteins were expressed in E. coli and purified using Pierce Anti-DYKDDDDK Magnetic Agarose, Sigma-Aldrich® Anti-FLAG™ M2 Magnetic Beads, and MBL Anti-DDDDK-tag mAb-Magnetic Agarose. Tagged protein was competitively eluted with Pierce 3x DYKDDDDK Peptide, and the results were analyzed by SDS-PAGE(A) and densitometry using the Invitrogen iBright Imaging System(B). Comparison between the starting lysate and elution fractions shows effective immunoprecipitation and elution of DYKDDDDK-tagged protein, with minimal background from the Pierce magnetic agarose compared to the other suppliers’ products.

Protein purification product features:

• Broad product selection—strong ion exchange and affinity supports for the purification and enrichment of proteins and antibodies; affinity ligands enable 1-step purification of recombinant and biotinylated proteins, while activated supports provide a platform for custom protein immobilization
• High performance—resins are designed to maximize protein yield and reduce background
• More formats—magnetic beads, loose resins, FPLC cartridges, and 96-well filter plates enable protein purification from screening and small-scale phases to process-scale purification
• Economical—pricing that is similar to or better than other leading suppliers

Types of affinity purificationDescriptionProtein immobilization
(Activated supports for custom immobilization)Uses activated supports and accessories for the immobilization of proteins, antibodies, and other molecules. These resins or magnetic beads are available separately or in convenient kits. Different reactive chemistries are available to optimize immobilization based on the ligand properties.

See covalent immobilization of affinity ligands learning resourceAntibody purificationProteins A, G, A/G, and L have unique properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). These ligands enable purification of general immunoglobulins from a crude sample. Depending on the sample source, an antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2–5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.

See antibody purification learning resourceRecombinant protein purification
(Fusion protein purification)Uses resins for the purification of recombinant proteins from cultures such as E. coli or Pichia. These resins are available in multiple formats to accommodate a variety of needs, from high-throughput screening to batch and pilot-scale purification. Superflow resins have undergone extensive chemical characterization. We have ligands targeting a variety of fusion tags, including 6xHis, GST, anti-DYKDDDDK (anti-FLAG™), c-Myc, and HA.

See fusion protein learning resourcePurification using magnetic AgaroseMagnetic agarose beads consist of highly crosslinked agarose encapsulating a ferrimagnetic core. The beads are 10–40 uM in size and have higher binding capacity than traditional magnetic beads. We offer a variety of ligands for immunoprecipitation (IP), co-immunoprecipitation (co-IP), pull-down, and other high throughput affinity screening applications, utilizing immobilized Protein A/G, Ni-NTA, Glutathione, and Anti-DYKDDDDK. The beads are removed from the solution manually using a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex Magnetic Particle Processor. Automated instruments are especially useful for higher throughput purification and screening of purification conditions.

See scientific poster: High capacity magnetic supports for automated antibody and epitope-tagged protein purificationsBiotin affinity purificationUses resins for the purification of biotinylated or desthiobiotinylated proteins, peptides, and other molecules. These resins are available in multiple pack sizes, as well as in spin columns, kits, FPLC cartridges, and coated plates. Different biotin-binding ligands are available based on elution conditions or level of purity.

See avidin-biotin interaction learning resourceImmunoprecipitation (IP)Small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic beads or agarose resin. IP is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

See immunoprecipitation learning resource
See IP supportCo-immunoprecipitation (co-IP) and pull-downSimilar to IP, except the target antigen precipitated by the antibody is used to co-precipitate its binding partner(s) or associated protein complex from the lysate and pull-downs. This method is used when antibodies to specific proteins are not available. These “bait” proteins are tagged with an epitope to which a high-affinity antibody is available and ectopically expressed in the cell of interest.

See co-immunoprecipitation learning resource
See pull-down assays learning resource
See co-IP and pull-down supportChIP, RIP, and protein-nucleic acid pull-downChromatin immunoprecipitation (ChIP) assays are performed to identify regions of the genome with which DNA-binding proteins, such as transcription factors and histones, associate. In ChIP assays, proteins bound to DNA are temporarily crosslinked and the DNA is sheared prior to cell lysis. The target proteins are immunoprecipitated along with the crosslinked nucleotide sequences, and the DNA is then removed and identified by PCR, sequenced, applied to microarrays, or analyzed in some other way.

RNA immunoprecipitation (RIP) uses an approach similar to ChIP, except that RNA-binding proteins are immunoprecipitated instead of DNA-binding proteins. Immunoprecipitated RNAs can then be identified by RT-PCR and cDNA sequencing.

See ChIP learning resource
See protein-nucleic acid interaction supportEndotoxin detection and removal kitsEndotoxin is a type of pyrogen and is a component of the exterior cell wall of Gram-negative bacteria. It is an unwanted and toxic by-product of recombinant proteins purified from E coli. Therefore, its removal from protein samples is an important step for downstream applications.

See app note: Eliminate endotoxins from protein and antibody samples

Overview of ion exchange, affinity, and activated supports

ApplicationPurity levelLigand and/or chemistryBase bead typePackaging optionsIon exchange purificationMedium to high (application-specific)Strong anion exchangePOROSLoose resinStrong cation exchangeAntibody purificationHighProtein A, protein G, protein A/GAgarose, magnetic beads, magnetic agarose, POROSLoose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin platesProtein LAgarose, magnetic beadsMelon GelAgaroseFusion protein purificationHighNi-NTA, Ni-IDA, high capacity EDTA compatible Ni-IMAC, cobalt, glutathioneAgarose, Superflow, magnetic beads, magnetic agaroseLoose resins or beads, spin columns and kits, chromatography cartridges, 96-well spin platesAnti-c-Myc, anti-HA, anti-FLAGAgarose, UltraLink magnetic beadsLoose resins or beads, kitsBiotin affinity purificationHighAvidin, streptavidin, NeutrAvidin, monomeric avidinAgarose, magnetic beadsLoose resins, spin columns and kits, chromatography cartridges, 96-well spin platesProtein immobilizationHighAmine-reactive, sulfhydryl-reactive, carbonyl-reactive, carboxyl-reactiveAgaroseLoose resins or dry powderEpoxy, tosyl-activated, carboxylic acid, amineMagnetic beadsLoose beads

Select your resin based on purification scale and application

ScaleHigh-throughput screeningHigh-throughput batchBatchPilotProcessDescriptionSmall scale, automation compatibleLab or bench scaleLab or bench scaleScale-up desiredProduction scaleYieldMicrogramMilligramMilligramGramKilogramFormatMagnetic particle processorMagnetic particle processor, 96-well spin plate (agarose)Gravity flow, spin column (agarose), fast protein liquid chromatography (FPLC) at low flow ratesFPLC at medium flow ratesFPLC at high flow ratesApplicationHigh-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysisHigh-throughput screening, interaction studies (IP, co-IP, pull-down), mutational analysis requiring mg scaleFunctional assays, structural analysisStructural analysis, intermediate-scale productionBulk productionRecommended resin typeMagnetic bead (1-2.8 µm)Magnetic agarose (10-40 µm)Agarose (45-165 µm)Superflow (45-165 µm)UltraLink resin (50-80 µm)POROS resin (50 µm)

Tips

The process of protein purification varies depending on the downstream analyses to be performed. Certain steps may be repeated or omitted to achieve the desired result.

Purification products

Protein immobilization

Product selection guidesProductsProtein immobilizationPierce NHS-Activated Agarose
AminoLink Coupling Resin
AminoLink Plus Coupling Resin
SulfoLink Coupling Resin
GlycoLink Coupling Resin
CarboxyLink CouplingAdditional activated supports and accessoriesPierce NHS-Activated Agarose Spin Columns
AminoLink Plus Immobilization Kit
AminoLink Plus Micro Immobilization Kit
AminoLink Immobilization Kit
AminoLink Reductant
Pierce CDI-activated Agarose Resin
SulfoLink Immobilization Kit for Peptides
UltraLink Iodoacetyl Micro Peptide Coupling Kit
GlycoLink Immobilization Kit
GlycoLink Micro Immobilization Kit
CarboxyLink Immobilization Kit

Antibody purification

Product selection guidesProductsAntibody purificationProtein AMagnetic beadsPierce Protein A Magnetic Beads
Dynabeads Protein A Magnetic BeadsResinsPierce Protein A Agarose
Pierce Protein A Plus Agarose
Pierce Recombinant Protein A Agarose
POROS MabCapture A SelectSpin columns and kitsNAb Protein A Plus Spin Columns
NAb Protein A Plus Spin KitsBuffersPierce Protein A IgG Binding BufferProtein GMagnetic beadsPierce Protein G Magnetic Beads
Dynabeads Protein G Magnetic BeadsResinsPierce Protein G Agarose
Pierce Protein G Plus Agarose
POROS MabCapture G SelectSpin columns and kitsNAb Protein G Spin Columns
NAb Protein G Spin Kits
Pierce Recombinant Protein GBufferPierce Protein G IgG Binding BufferProtein A/GMagnetic beadsPierce Protein A/G Magnetic Agarose Beads
Pierce Protein A/G Magnetic BeadsResinsPierce Protein A/G Agarose
Pierce Protein A/G Plus Agarose
POROS MabCapture A/G SelectSpin columns and kitsNAb Protein A/G Spin Columns
NAb Protein A/G Spin Kit
Pierce Recombinant Protein A/GBufferPierce Protein A/G IgG Binding BufferProtein LMagnetic beadsPierce Protein L Magnetic BeadsResinsPierce Protein L Agarose
Pierce Protein L Plus AgaroseSpin columns and kitsNAb Protein L Spin Columns
NAb Protein L Spin Kit
Pierce Recombinant Protein LMelon GelMelon Gel IgG Spin Purification Kit
Melon Gel IgG Purification Kit
Melon Gel Monoclonal IgG Purification Kit
Monoclonal IgG Purification Kit
Melon Gel Spin Plate Kit for IgG Screening
Melon Gel Chromatography Cartridges (1 mL or 5 mL)
Melon Gel Regenerant
Ascites Conditioning Reagent for Melon Gel
Melon Gel Purification BufferBuffers and related productsPierce IgG Elution Buffer
Pierce Gentle Ag/Ab Binding Buffer, pH 8.0
Pierce Gentle Ag/Ab Elution Buffer, pH 6.6
Pierce Gentle Ag/Ab Binding and Elution Buffer Kit
Pierce Saturated Ammonium Sulfate Solution
Pierce Thiophilic Adsorption Kit
Pierce Mannan Binding Protein Agarose
Pierce IgM Purification Kit
Elution Buffer for Pierce IgM Purification Kit
MBP Column Preparation Buffer for Pierce IgM Purification Kit
Pierce Jacalin Agarose

Recombinant protein purification

Product selection guidesProductsHis-tagged (6xHis) protein purificationPierce High-Capacity Ni-IMAC MagBeads, EDTA Compatible
Pierce High-Capacity Ni-IMAC Resin, EDTA Compatible
Pierce Ni-NTA Magnetic Beads
Pierce Ni-NTA Magnetic Agarose Beads
HisPur Ni-NTA Agarose Resin        
HisPur Ni-NTA Superflow Resin
HisPur Cobalt Agarose Resin
HisPur Cobalt Superflow ResinGST-tagged protein purificationPierce Glutathione Magnetic Agarose Beads
Pierce Glutathione Agarose Resin
Pierce Glutathione Superflow ResinOther epitope-tagged protein purificationPierce Anti-DYKD4K (FLAG) Magnetic Agarose
Pierce Anti-DYKD4K (FLAG) Affinity Resin UltraLink
Pierce Anti-c-Myc Magnetic Beads
Pierce Anti-c-Myc Agarose (Superflow 6)
Pierce Anti-HA Magnetic Beads
Pierce Anti-HA AgarosePurification using magnetic agarosePierce Ni-NTA Magnetic Agarose Beads
Pierce Glutathione Magnetic Agarose Beads
Pierce Anti-DYKD4K (FLAG) Magnetic Agarose Beads

Biotin affinity purification

Product selection guidesProductsBiotin affinity purificationMagnetic beadsPierce Streptavidin Magnetic BeadsResinsPierce Avidin Agarose
Pierce Streptavidin Agarose
Pierce Streptavidin Agarose HC
Pierce NeutrAvidin Agarose
Pierce NeutrAvidin Agarose HC
Pierce Monomeric Avidin AgaroseRelated products for biotin-binding applicationsInvitrogen CaptAvidin Agarose (Sedimented Bead Suspension)
Pierce Streptavidin Agarose Columns
Pierce High Capacity Streptavidin Chromatography Cartridge
Pierce Monomeric Avidin Agarose Kit
Pierce Biotin
Pierce Iminobiotin Agarose

IP, co-IP, pull-down

Product selection guidesProductsImmunoprecipitation (IP)Antibody-binding IP productsProtein A, G, A/GMagnetic beads

• Dynabeads Protein A
• Dynabeads Protein G
• Pierce Protein A/G

Kits

• Dynabeads Protein A kit
• Dynabeads Protein G kit
• Pierce Protein A/G kit
• Pierce Crosslink Kit (A/G)

Secondary antibodies (anti-mouse, anti-rabbit)Magnetic beads

• Dynabeads M-280, Sheep-anti Mouse IgG
• Dynabeads M-280, Sheep-anti Rabbit IgG

Surface-activated beads (epoxy)Kits

• Dynabeads Antibody Coupling Kit
• Dynabeads Co- Immunoprecipitation Kit
• See more choices in surface-activated Dynabeads

Biotin-binding IP products

• Pierce Streptavidin Magnetic Beads
• Dynabeads M-280 Streptavidin
• Dynabeads M-270 Streptavidin
• Dynabeads MyOne Streptavidin C1
• Dynabeads MyOne Streptavidin T1
• DynaMag-2 Magnet

Recombinant protein (fusion tag) IP products

• HisPur Ni-NTA Magnetic Beads
• Pierce Ni-NTA Magnetic
• Agarose Beads
• Dynabeads His-Tag Isolation and Pulldown
• Pierce Glutathione Magnetic Agarose Beads
• Pierce Anti-DYKDDDDK Magnetic Agarose
• Pierce Anti-HA Magnetic Beads
• Pierce HA-Tag Magnetic IP/Co-IP Kit
• Pierce Anti-c-Myc Magnetic Beads
• Pierce c-Myc-Tag Magnetic IP/Co-IP Kit
• DynaMag-2 Magnet

Co-immunoprecipitation (co-IP) and pull-downSpin column

• Pierce Direct IP Kit
• Pierce Crosslink IP Kit
• Pierce Co-IP Kit (agarose)

Magnetic beads

• Dynabeads Co-IP Kit
• Dynabeads His-Tag Isolation and Pulldown Kit

ChIP, RIP, and Protein-Nucleic Acid Pull-DownChIP

• MAGnify Chromatin Immunoprecipitation System
• Pierce Magnetic ChIP Kit
• Pierce ChIP-grade Protein A/G Magnetic Beads
• Proteinase K Solution, ChIP grade

RIP

• Pierce Magnetic RNA-Protein Pull-Down Kit
• Pierce Streptavidin Magnetic Beads
• Pierce RNA 3' End Desthiobiotinylation Kit

Ion exchange purification

Product selection guidesProductsIon exchange purificationIon exchange spin columnsPierce (SCX) Spin Columns
Pierce (SAX) Spin Columns
Ion Exchange PurificationIon exchange resinsPOROS XS Resin
POROS XQ Resin
POROS 50 HQ Resin

Endotoxin removal

Product selection guidesProductsEndotoxin detectionPierce Chromogenic Endotoxin Quant Kit
Pierce LAL Chromogenic Endotoxin Quantitation KitEndotoxin removalLoose resin
Spin columns (0.25 mL, 0.5 mL, or 1 mL)

Clean up your protein sample

Many detergents and salts used in protein extraction formulations may have adverse effects on protein function or stability, or may interfere with downstream analysis. Therefore, it may be necessary to remove or reduce these contaminants following cell lysis or subsequent sample processing such as protein purification.

Download Protein Clean-Up Technical Handbook

Discover more about protein dialysis, desalting, and concentration

Tips

• If the protein concentration is too dilute for further processing or analysis, the sample can be concentrated quickly using centrifugal concentrators.
• For buffer exchange during concentration technique, the retentate can be diluted with exchange buffer and centrifuged. This process can be repeated until the desired level of exchange or desalting has been achieved.

Protein clean-up products

Protein dialysis

Dialysis is a classic clean-up technique that removes small molecules and unwanted compounds by selective diffusion through a semi-permeable membrane. A sample and a buffer solution are placed on opposite sides of the membrane. Proteins that are larger than the membrane pores are retained on the sample side of the membrane, but smaller molecules (contaminants) diffuse freely through the membrane until an equilibrium concentration is achieved. Through this technique, the concentration of small contaminants in the sample can be decreased to acceptable levels.

Protein dialysis products features:

• Excellent sample recovery—low-binding plastics and membranes help minimize sample loss compared to filtration and resin systems
• Convenience—easy-to-grip design helps simplify sample addition and removal with syringe and/or pipette
• Secure—sealed membranes help prevent leakage that can occur with dialysis tubing and homemade devices
• Validated—each device is be leak-tested during production

Discover more about protein dialysis
See protein dialysis learning resource

Product highlight

MWCO* membrane10-100 µL
Pierce 96-well Microanalysis Plate10-2,000 µL
Slide-A-Lyzer MINI Dialysis Device0.1-70 mL
Slide-A-Lyzer G2 Dialysis Cassette0.1-30mL
Slide-A-Lyzer Dialysis Cassette150-250 mL
Slide-A-Lyzer Dialysis Flask15-100 mL
SnakeSkin Dialysis Tubing2KN/A✔✔✔✔
See productN/A3.5K✔
See product✔✔✔✔
See product✔7KN/A✔✔✔N/A✔10K✔
See product✔✔✔✔
See product✔20KN/A✔✔✔✔
See productN/ASee full comparison of these products ›

*MWCO: Molecular weight cut off.

Protein recovery by molecular weight cutoff (MWCO)

Sample retention by the 2K, 3.5K, 7K, 10K, and 20K MWCO Thermo Scientific Slide-A-Lyzer cassette membrane. Individual proteins or vitamin B12 (1 mg/mL) in either saline or 0.2 M carbonate-bicarbonate buffer, pH 9.4 were dialyzed overnight (17 hours) at 4°C. The amount of retentate was estimated using either the Pierce BCA Protein Assay Kit or absorption at 360 nm (for vitamin B12).

Dialysis rates for various formats

The rate of removal of NaCl using various dialysis products. NaCl removal from samples was determined by measuring the conductivity of the retentate at the indicated times. (A)Slide-A-Lyzer MINI Dialysis Device (10K MWCO, 2 mL) versus conventional dialysis. Bovine serum albumin (BSA) samples (2 mL, 0.25 mg/mL in 1 M NaCl) were dialyzed against 45 mL of water in 50 mL disposable conical tubes on an orbital shaker (300 rpm) at room temperature. The water was changed once after 2 hours. Results are the average of two samples. For conventional dialysis, the samples were dialyzed against 2 L of water in a beaker with stirring. Greater than 95% of NaCl was removed within 4 hours. (B) Samples of 0.1 mL (0.4 mg/mL cytochrome C containing 1 M NaCl) were dialyzed in the Pierce 96-well Microdialysis Plate against 1.8 mL of water at RT with gentle shaking. The buffer was changed at 1-, 2-, and 3-hour intervals over a 4-hour period. Removal of NaCl was >83% after 2 hours and >99% after 4 hours. (C) Proteins in 200 mL samples containing 1 M NaCl were dialyzed at room temperature using Slide-A-Lyzer Dialysis Flasks with 2K, 3.5K, 10K, and 20K MWCOs. The dialysis buffer (4 L) was changed after 2 and 5 hours (triangles; also at 41 hours for the 2K condition). Greater than 95% of NaCl was removed within 8 to 18 hours (41 hours for the 2K condition).

Protein desalting

Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. Using this technique, a resin is selected with pores large enough for salts to penetrate, but small for the protein of interest to enter. This causes contaminants to slow down their rate of migration. The larger and faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.

Discover more about protein desalting
See protein desalting learning resource

Protein desalting products features:

• High performance—proprietary resin enables excellent protein recovery and efficient contaminant removal
• Flexible—available in spin columns, filter spin plates, and cartridges for a range of needs
• Fast—no fraction screening or waiting for protein to emerge by gravity flow
• Economical—cost-effective products that offer great performance

Product highlight

TypeSpin columnsSpin platesChromatography columnsFormatMicro0.5 mL2 mL5 mL10 mL96-well1 mL5 mLResin bed75 µL0.5 mL2 mL5 mL10 mL550 µL1 mL5 mLSample volume (7K MWCO)2-12 µL30-130 µL200-700 µL500-2,000 µL700-4,000 µL20-100 µL50-250 µL100-1,500 µLSample volume (40K MWCO)5-14 µL70-200 µL200-900 µL300-2,000 µL1,000-4,000 µL20-100 µLN/AN/ASee full comparison of these products ›

Comparison of protein recovery and sample dilution

Zeba Spin Desalting Columns result in a high protein recovery while providing minimal sample dilution over a wider range of sample concentrations and volumes compared to alternative products.Zeba Spin Desalting Columns, 10 mL (7K MWCO) and GE PD-10 Columns were used to desalt 1.5, 2.5, and 3.5 mL BSA samples at a concentration of 0.04, 0.2, and 1 mg/mL. Desalting was performed according to the manufacturers’ recommended protocols; both the spin and gravity protocols were used for the GE PD-10. Protein recovery was analyzed by SDS-PAGE. For each electrophoresis gel, an aliquot of starting sample equal to 1 μg of BSA was loaded in lane 1 as the loading control; all other desalted samples were loaded in the gel at the same volume as the loading control. Differences in intensity between lanes are a combination of protein recovery and sample dilution caused by desalting. The largest differences in recovery and concentration are noted in the highlighted area.

Protein concentrators

Protein concentration is similar to dialysis and uses a semi-permeable membrane to separate proteins from low molecular weight compounds. Unlike dialysis, which relies on passive diffusion, concentration is achieved by forcing solution through membrane using centrifugation. During centrifugation, both buffer and low molecular weight solutes are forced through the membrane where they are collected on the other side (filtrate). Macromolecules (proteins) remain on the sample side of the membrane, where they become concentrated to a smaller volume (retentate), as the reagent is forced across the membrane to the other side.

Discover more about protein concentrators
See protein dialysis, desalting, and buffer exchange learning resource

Protein concentrator product features

• Rapid processing—unique design minimizes membrane fouling; and sample concentration of 10- to 30-fold can be achieved in 5–30 minutes for 10K MWCO (device-dependent times may vary for other MWCOs), even with particle-laden solutions
• High recovery—retain >90% of protein samples while removing contaminants or exchanging buffers
• Convenient—clear markings, wide sample chamber, and removable filtrate chamber make handling simple and easy
• Instrument compatible—can be used with standard centrifuges utilizing either fixed-angle or swinging-bucket rotors

Product highlight

Volume range0.1–0.5 mL2–6 mL5–20 mL20–100 mLMWCOs available3K, 10K, 30K, 100K3K, 10K, 30K, 100K3K, 10K, 30K, 100K5K, 10K, 30K, 100KProcessing time*3-15 min15-90 min15-60 min15-90 minRetentate volume range*9-67 µL51-174 µL121-777 µL1.9-3.5 mLProtein recovery range*95-100%94-100%94-100%92-98%See full comparison of these products ›

*Four different protein solutions were used for each MWCO

Comparison of protein recovery between Pierce Protein Concentrators (using 3K, 5K, 10K, 30K, or 100K MWCO) and other vendors for 0.5 mL, 6 mL, 20 mL, and 100 mL concentrators. Samples of different protein solutions were centrifuged in Pierce Protein Concentrators and other suppliers’ concentrators according to manufacturers’ instructions: 0.5 mL (15,000 x g), 5 mL (4,000 x g), 20 mL (4,700 x g), and 100 mL (1,200 x g). Samples were centrifuged until a greater than 15- to 30-fold decrease in sample volume was achieved; protein concentration was measured by either Pierce BCA Protein Assay Kit (0.5 mL concentrators only) or absorbance at A280.

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