5 Steps to Protein Isolation and Purification

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.

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

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.

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

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

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.
   

Discover more about cell lysis and fractionation
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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.

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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

Download protein purification poster

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

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.

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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.

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

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See protein dialysis learning resource

*MWCO: Molecular weight cut off.

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).

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).

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.

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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

Download Zeba Desalting Handbook

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.

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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

*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.

Total protein quantitation

Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use colorimetric or fluorescent detection with fluorometers, spectrophotometers, or plate readers. Every protein assay has limitations depending on the application and the specific protein sample analyzed. The most useful features to consider when choosing a protein assay are sensitivity (lower detection limit), compatibility with common substances in samples (e.g., detergents, reducing agents, chaotropic agents, inhibitors, salts, and buffers), standard curve linearity, and protein-to-protein variation.

Colorimetric protein assays

Colorimetric signals can be detected using a microplate reader or spectrophotometer. The most popular colorimetric protein assays are:

• BCA Assays—Protein-copper chelation with secondary detection of the reduced copper.
• Bradford Assays—Protein-dye binding with direct detection of the color change associated with the bound dye.
   

Fluorescent protein assays

Fluorescence-based protein quantitation is an alternative to colorimetric methods. Fluorescence detection methods offer excellent sensitivity, requiring less protein sample thereby leaving more sample available for your experiment. Additionally, read time is not a critical factor, so the assays can be readily adapted for automated high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader.

Discover more about protein assays and analysis
See protein quantitation and detection learning resource
Learn more about methods and technologies for identifying and measuring proteins

Use the interactive Protein Assay Selection Guide

Tips

• No one reagent is the ideal or best protein assay method. Each method has its advantages and disadvantages.
  Use the interactive Protein Assay Selection Guide to filter products based on sample type, assay time, read-out (colorimetric or fluorescent), and compatibility to detergents or reducing agents
• To learn about the different ways to quantitate protein concentrations continue reading the article: Overview of protein assays.

*With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate

Search ELISA kits  Explore western blot products

Tips

• Western blotting tips & tricks
• Sample prep for MS analysis

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