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5 Steps to Fundamental Protein Preparation
The goal of protein preparation is to generate quality protein samples that maximize the chance of a successful downstream application (ex. western blotting, ELISA, immunoprecipitation, mass spectrometry). Because proteins are diverse in both structure and in function, there are often challenges with balancing efficient extraction and maintaining protein function required for the downstream analysis. This balance is crucial for studies to examine biological phenomena, and success is dependent on the methods and reagents required for protein preparation.
Featured webinar
Protein sample prep strategies
This webinar will focus on different membrane protein solubilization strategies and what enrichment or purification is necessary depending upon the downstream mode of detection or analysis.
Follow these 5 steps to obtain optimal protein preparation conditions
Extract your target protein from the sample
The first step in protein analysis is cellular extraction, which requires liberation of protein from the sample source. Whether using mechanical or detergent-based extraction methods, this process inevitably disrupts cellular homeostasis and contributes to the degradation or destabilization of proteins. Therefore, the quality of the data obtained from protein samples directly depends upon the integrity of the protein during the extraction process. For example, some extraction methods may be efficient at cell lysis and solubilization of cell contents but are protein-denaturing, thereby preventing detection and analysis of native protein interactions.
Sample type
Cultured mammalian cells, mammalian tissues, and primary cells are frequently used as sources of physiologically relevant endogenous proteins (including post-translational modifications) as well as overexpression systems (transient or stable). When extracting protein from mammalian tissues, some gentle means of enzymatic and/or mechanical disruption is required to help separate cells from the more complex tissue matrix. For cultured mammalian cells and primary cells that only have a plasma membrane separating the cell contents from the environment, reagents containing detergents can disrupt the protein-lipid membrane bilayer, making total protein extraction relatively easy. Other organisms that are commonly studied or used for recombinant protein expression systems include bacteria, yeast, and plants. These cell types contain cell walls that require additional enzymatic or mechanical disruption to efficiently release their protein content. However, detergent-based solutions have been developed to effectively extract and solubilize protein from these cells without the need for mechanical disruption, which is especially important when processing higher sample numbers or when automating extraction and purification protocols.
Sub-cellular fractionation
For most studies, generating whole cell lysates is an easy, straightforward way to prepare a soluble protein sample for direct detection or for further purification or fractionation. However, the yield or enrichment of a specific protein can be improved significantly if the cell is fractionated into different compartments or organelles before protein extraction. Mechanical lysis usually disrupts all cellular compartments, thereby making it difficult to isolate specific cellular fractions. However, by carefully optimizing reagents, stepwise differential detergent procedures have been developed to separate nuclear, cytosolic, and membrane protein fractions. With this strategic approach, hydrophobic membrane proteins can be solubilized and separated from the hydrophilic proteins, and intact nuclei, mitochondria, and other organelles can be isolated for direct study or separate protein extraction.
Figure 1. Source of specimen or sample type. Suitable protein sources for sample collection may include native sources derived from mammalian cell cultures, tissues, or body fluids. Alternatively, proteins may be over expressed in model systems such as yeast, bacteria, insect, or mammalian cells.
Figure 2. Protein preparation decision tree. This illustration depicts many of the factors involved in selecting a particular protein isolation strategy.
Figure 3. Protein sample preparation. The workflow begins with cell lysis, and the steps involved in sample preparation are designed to move the protein containing fraction from the initial biological source into a homogeneous solution, while preserving the in vivo state of the protein. Obtaining high quality lysates is critical for the successful outcome of downstream applications.
| Product highlight | Description |
|---|---|
| Total protein extraction reagents (PERs) | Protein extraction reagents designed for whole cell extraction from a variety of sample types |
| Fractionation reagents | Reagents designed to enrich or fractionate for cellular organelles including nuclear, cytosolic, mitochondrial, membrane, plasma membrane, and extracellular matrix compartments |
| GPCR Extraction and Stabilization Reagent | Protein extraction reagent for efficient solubilization and stabilization of GPCRs |
| Surfact-Amps detergents | Detergent solutions ideal for applications or assays that are sensitive to contaminants present in unpurified detergents |
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.
- 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 products:
Preserve your target protein
Cell lysis disrupts cell membranes and organelles, resulting in proteolytic activity that can reduce protein yield and function. To prevent degradation of extracted proteins and maintain their activity, protease and phosphatase inhibitors are frequently added to lysis reagents.
Protease inhibitors function by binding to protease active sites. Due to the differences in the proteolytic mechanisms, no single compound can effectively inhibit all proteases, and therefore, a mixture or cocktail of several different inhibitor compounds is needed to ensure that protein extracts do not degrade before downstream analyses. Typical cocktails include small molecule inhibitors of serine, cysteine, and aspartic acid proteases as well as aminopeptidases and metalloproteases. While some inhibitors are irreversible, many are reversible and require their continued presence in the crude sample until further purification removes the threat of proteolytic activity.
Likewise, phosphatases vary so an effective phosphatase cocktail containing inhibitors for serine, threonine, tyrosine, acidic, and alkaline phosphatases is recommended to preserve fragile phosphorylation post-translational modifications.
Preservation method(s) should be used to prevent your target protein from degradation:
- Work quickly and keep samples cold (consider freezing samples in liquid nitrogen)
- Inhibit or inactivate endogenous proteases and phosphatases
- Add protective or stabilizing compounds, such as reducing agents and enzyme inhibitors
- Stabilize or inactivate proteins by precipitation
| Product highlight | Description |
|---|---|
| Protease and Phosphatase Inhibitors | Broad-spectrum protease and phosphatase inhibition in liquid and tablet formats. |
Tips
- Most researchers use a mixture of several different inhibitor compounds to ensure the protein extracts do not degrade before analysis of target interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
Protein preservation products:
Clean up your protein sample
After protein extraction, the protein samples often contain contaminants that are not compatible with protein stability or downstream applications. Dialysis, desalting, and diafiltration (concentration) are three common methods used to remove common contaminants, such as salts and detergents, from protein samples. Depending on the end application requirements, considerations for method choice may include amount of sample input, requirement for functional protein, and processing time. There are a variety of options and formats available for dialysis, desalting, and diafiltration methods.
Dialysis
Dialysis is a classic separation technique that removes small molecules and unwanted compounds from protein in solution by way of 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.
Desalting
Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. In this technique a resin is selected with pores large enough for salts to penetrate but small enough for the protein of interest to enter. This causes contaminants to slow down their rate of migration, and the larger faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.
Concentration
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 by 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.
| Product highlight | Category | Description | Sample volume |
|---|---|---|---|
| Slide-A-Lyzer products | Dialysis | Remove salts or other small molecular weight contaminants | 10 uL to 250 mL |
| Zeba products | Desalting | Removes salt | 2 uL to 4 mL |
| Dye and biotin removal products | Small molecule clean up | Remove free/unused dye, biotin, crosslinkers, and reducing agents | 40 uL to 4mL |
| Concentrators | Concentration | Concentration, desalting, buffer exchange | 100 uL to 100 mL |
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.
Discover more about protein dialysis, desalting, and concentration
Protein clean-up products:
| Product category | Products |
|---|---|
| Dialysis | Slide-A-Lyzer products |
| Small molecule removal | Biotin, dye, crosslinkers, and reducing agent removal products |
| Desalting | Zeba products |
| Concentration | Protein concentrators |
| Detergent removal products | HiPPR Detergent Removal 96-well Spin Plates |
| Abundant protein depletion | High Select Top 14 and Top 2 reagents |
Quantify your target protein
Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use fluorescent or colorimetric 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 provide 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.
| Product highlight | Category | Description |
|---|---|---|
| Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards | Colorimetric | Two-component, high-precision, detergent-compatible protein assay. Compared to most dye-binding methods, the BCA assay is affected much less by protein compositional differences, providing greater concentration accuracy. The Dilution-Free BSA Protein Standards, included in the kits, eliminate the need for serial dilutions. |
| Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit | Colorimetric | This kit maintains the key characteristics of the traditional BCA assay but eliminates sample and standard curve dilution steps. Additionally, it only requires a 5-minute, room temperature incubation, similar to dye-binding methods. |
| Qubit Protein Broad Range (BR) Assay Kit | Fluorescence | This fluorescent protein assay provides accurate protein quantification across a broad range (0.1 – 20 mg/mL), and is specifically designed for the Qubit 4 Fluorometer. It is compatible with both reducing agents and detergents. |
| Qubit 4 Fluorometer | Fluorometer | The Qubit 4 Fluorometer is the latest version of the popular Qubit fluorometer designed to accurately measure protein quantity. |
Tips
- No one reagent is the ideal or best method for all applications. 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.
Find assays with Protein Assay Selection Guide Download Protein Assay Technical Handbook
Recommended assays based on common lysis buffers
| Lysis/Extraction buffer | Recommended protein assay | If adding reducing agents or metal chelators (e.g., DTT above 1 mM, EDTA above 10mM) |
|---|---|---|
| RIPA | BCA Protein Assay | BCA Protein Assay-Reducing Agent Compatible |
| M-PER Mammalian Protein Extraction Reagent | BCA Protein Assay | Bradford Assay |
| B-PER Bacterial Protein Extraction Reagents | BCA Protein Assay | BCA Protein Assay-Reducing Agent Compatible |
| NE-PER Nuclear and Cytoplasmic Extraction Reagents | BCA Protein Assay | Bradford Assay |
| Cell Extraction Buffer | BCA Protein Assay - Reducing Agent Compatible |
Tips
- BCA Protein Assays have a unique advantage over Bradford assays, as they are compatible with samples that contain up to 5% surfactants (detergents) and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy.
Recommended assay based on required sensitivity
For quantitation of low volume or very dilute samples, fluorescent assays can provide working ranges down to 10 ng/ml compared to enhanced colorimetric assays at 500 ng/mL and 2,000 ng/ml for standard colorimetric protocols.
| Sample type requirements | Recommended protein assay |
|---|---|
| Very dilute or small sample volumes (minimum sample volume: 1 µL) | CBQCA Protein Quantitation Kit |
Tips
- The CBQCA Protein Quantitation Kit is a very sensitive assay for quantitating proteins in solution, capable of detecting as low as 10 ng of protein per mL. Similar in sensitivity to our NanoOrange protein quantitation reagent (N-6666), CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions, or detergents, and for lipoproteins and small peptides.
Recommended equipment
Once the total protein assay is complete, results can be read using either a UV-Vis spectrophotometer for colorimetric based assays or a fluorometer for fluorescence-based assays.
Multiskan Sky Microplate Spectrophotometer
The Multiskan Sky Microplate Spectrophotometer is a UV/Vis microplate spectrophotometer designed to be convenient and easy to use for virtually any photometric research application, especially DNA, RNA, and protein analysis. It is ideal for multi-user environments where a variety of endpoint, kinetic, and spectral assays are performed. Multiskan Sky instruments are available in three different configurations. Touch screen models offer the flexibility to use the stand-alone instrument or in conjunction with Thermo Scientific SkanIt PC software. The Multiskan Sky model operated solely via SkanIt software is ideal for users who rely on a PC for all operations. Cuvette reading capability is also offered in some models.
Protein quantitation products:
References
- Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
- Gornall, A. G. (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766.
- Lowry, O. H., et al. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
- Smith, P. K., et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.
- Stoscheck, C. M. (1987) Protein assay sensitive at nanogram levels. Anal Biochem 160:301–305.
Detect and measure your target protein
There are various methods that can be used to detect and measure your target protein depending on your experimental needs. Below are common techniques used to detect and measure proteins from complex mixtures (e.g., lysates, sera) and the typical requirements for each.
| ELISA | Western Blotting | Mass Spec | |
|---|---|---|---|
| Advantages |
|
|
|
| Sensitivity | <5–10 pg/mL | Low femtogram to high attogram* | Attomolar range (1018) |
| Lysis buffer compatibility |
|
|
|
| Typical total protein required | 0.1–1 µg/mL | 1–50 µg | <1 µg |
| Equipment required | Plate reader | X-ray film or CCD imaging equipment | Mass spectrometer |
| *With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate | |||
Recommended equipment
ELISA measurement and analysis
Multiskan Sky Microplate Spectrophotometer
In addition to reliable ELISA measurements, perform UV-Vis photometric research applications such as DNA, RNA, and protein analysis with the Thermo Scientific Multiskan Sky Microplate Spectrophotometer. The Multiskan Sky reader features a broad wavelength range (200–1000 nm) path length correction and a fast reading speed. Its intuitive touchscreen user interface, on-board software, and built-in protocols let you run quick measurements directly from the instrument. Alternatively, with any instrument purchase you can use our unlimited license for our easy-to-use Thermo Scientific SkanIt Software, with access to our extensive online library of ready-made protocols.
Western blot documentation and analysis
iBright Imaging Systems
The iBright 1500 Imaging Systems are powerful and easy-to-use, providing sensitive, streamlined, multimode image capture for gel and western blot documentation. The iBright FL1500 Imaging System is capable of easily capturing 4-plex images. It features a large capacitive touch-screen interface and intelligently designed software.
Protein detection products:
| Product/workflow highlights | Category | Description |
|---|---|---|
| Protein gels | Electrophoresis and Western Blotting | Unique protein gel chemistries for all application needs |
| iWestern workflow | Electrophoresis and Western Blotting | An intelligent, start to finish western blotting solution |
| Protein mass spectrometry workflow | Mass spectrometry | Mass spec reagents, instrumentation, and software solutions for proteomics workflows for discovery and targeted proteomics applications |
| ELISA Kits | ELISA | 5 simple steps to achieve consistent and accurate results when running ELISAs |
Tips
Discover more about western blotting, ELISA, and mass spec
Extract your target protein from the sample
The first step in protein analysis is cellular extraction, which requires liberation of protein from the sample source. Whether using mechanical or detergent-based extraction methods, this process inevitably disrupts cellular homeostasis and contributes to the degradation or destabilization of proteins. Therefore, the quality of the data obtained from protein samples directly depends upon the integrity of the protein during the extraction process. For example, some extraction methods may be efficient at cell lysis and solubilization of cell contents but are protein-denaturing, thereby preventing detection and analysis of native protein interactions.
Sample type
Cultured mammalian cells, mammalian tissues, and primary cells are frequently used as sources of physiologically relevant endogenous proteins (including post-translational modifications) as well as overexpression systems (transient or stable). When extracting protein from mammalian tissues, some gentle means of enzymatic and/or mechanical disruption is required to help separate cells from the more complex tissue matrix. For cultured mammalian cells and primary cells that only have a plasma membrane separating the cell contents from the environment, reagents containing detergents can disrupt the protein-lipid membrane bilayer, making total protein extraction relatively easy. Other organisms that are commonly studied or used for recombinant protein expression systems include bacteria, yeast, and plants. These cell types contain cell walls that require additional enzymatic or mechanical disruption to efficiently release their protein content. However, detergent-based solutions have been developed to effectively extract and solubilize protein from these cells without the need for mechanical disruption, which is especially important when processing higher sample numbers or when automating extraction and purification protocols.
Sub-cellular fractionation
For most studies, generating whole cell lysates is an easy, straightforward way to prepare a soluble protein sample for direct detection or for further purification or fractionation. However, the yield or enrichment of a specific protein can be improved significantly if the cell is fractionated into different compartments or organelles before protein extraction. Mechanical lysis usually disrupts all cellular compartments, thereby making it difficult to isolate specific cellular fractions. However, by carefully optimizing reagents, stepwise differential detergent procedures have been developed to separate nuclear, cytosolic, and membrane protein fractions. With this strategic approach, hydrophobic membrane proteins can be solubilized and separated from the hydrophilic proteins, and intact nuclei, mitochondria, and other organelles can be isolated for direct study or separate protein extraction.
Figure 1. Source of specimen or sample type. Suitable protein sources for sample collection may include native sources derived from mammalian cell cultures, tissues, or body fluids. Alternatively, proteins may be over expressed in model systems such as yeast, bacteria, insect, or mammalian cells.
Figure 2. Protein preparation decision tree. This illustration depicts many of the factors involved in selecting a particular protein isolation strategy.
Figure 3. Protein sample preparation. The workflow begins with cell lysis, and the steps involved in sample preparation are designed to move the protein containing fraction from the initial biological source into a homogeneous solution, while preserving the in vivo state of the protein. Obtaining high quality lysates is critical for the successful outcome of downstream applications.
| Product highlight | Description |
|---|---|
| Total protein extraction reagents (PERs) | Protein extraction reagents designed for whole cell extraction from a variety of sample types |
| Fractionation reagents | Reagents designed to enrich or fractionate for cellular organelles including nuclear, cytosolic, mitochondrial, membrane, plasma membrane, and extracellular matrix compartments |
| GPCR Extraction and Stabilization Reagent | Protein extraction reagent for efficient solubilization and stabilization of GPCRs |
| Surfact-Amps detergents | Detergent solutions ideal for applications or assays that are sensitive to contaminants present in unpurified detergents |
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.
- 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 products:
Preserve your target protein
Cell lysis disrupts cell membranes and organelles, resulting in proteolytic activity that can reduce protein yield and function. To prevent degradation of extracted proteins and maintain their activity, protease and phosphatase inhibitors are frequently added to lysis reagents.
Protease inhibitors function by binding to protease active sites. Due to the differences in the proteolytic mechanisms, no single compound can effectively inhibit all proteases, and therefore, a mixture or cocktail of several different inhibitor compounds is needed to ensure that protein extracts do not degrade before downstream analyses. Typical cocktails include small molecule inhibitors of serine, cysteine, and aspartic acid proteases as well as aminopeptidases and metalloproteases. While some inhibitors are irreversible, many are reversible and require their continued presence in the crude sample until further purification removes the threat of proteolytic activity.
Likewise, phosphatases vary so an effective phosphatase cocktail containing inhibitors for serine, threonine, tyrosine, acidic, and alkaline phosphatases is recommended to preserve fragile phosphorylation post-translational modifications.
Preservation method(s) should be used to prevent your target protein from degradation:
- Work quickly and keep samples cold (consider freezing samples in liquid nitrogen)
- Inhibit or inactivate endogenous proteases and phosphatases
- Add protective or stabilizing compounds, such as reducing agents and enzyme inhibitors
- Stabilize or inactivate proteins by precipitation
| Product highlight | Description |
|---|---|
| Protease and Phosphatase Inhibitors | Broad-spectrum protease and phosphatase inhibition in liquid and tablet formats. |
Tips
- Most researchers use a mixture of several different inhibitor compounds to ensure the protein extracts do not degrade before analysis of target interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
Protein preservation products:
Clean up your protein sample
After protein extraction, the protein samples often contain contaminants that are not compatible with protein stability or downstream applications. Dialysis, desalting, and diafiltration (concentration) are three common methods used to remove common contaminants, such as salts and detergents, from protein samples. Depending on the end application requirements, considerations for method choice may include amount of sample input, requirement for functional protein, and processing time. There are a variety of options and formats available for dialysis, desalting, and diafiltration methods.
Dialysis
Dialysis is a classic separation technique that removes small molecules and unwanted compounds from protein in solution by way of 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.
Desalting
Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. In this technique a resin is selected with pores large enough for salts to penetrate but small enough for the protein of interest to enter. This causes contaminants to slow down their rate of migration, and the larger faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.
Concentration
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 by 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.
| Product highlight | Category | Description | Sample volume |
|---|---|---|---|
| Slide-A-Lyzer products | Dialysis | Remove salts or other small molecular weight contaminants | 10 uL to 250 mL |
| Zeba products | Desalting | Removes salt | 2 uL to 4 mL |
| Dye and biotin removal products | Small molecule clean up | Remove free/unused dye, biotin, crosslinkers, and reducing agents | 40 uL to 4mL |
| Concentrators | Concentration | Concentration, desalting, buffer exchange | 100 uL to 100 mL |
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.
Discover more about protein dialysis, desalting, and concentration
Protein clean-up products:
| Product category | Products |
|---|---|
| Dialysis | Slide-A-Lyzer products |
| Small molecule removal | Biotin, dye, crosslinkers, and reducing agent removal products |
| Desalting | Zeba products |
| Concentration | Protein concentrators |
| Detergent removal products | HiPPR Detergent Removal 96-well Spin Plates |
| Abundant protein depletion | High Select Top 14 and Top 2 reagents |
Quantify your target protein
Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use fluorescent or colorimetric 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 provide 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.
| Product highlight | Category | Description |
|---|---|---|
| Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards | Colorimetric | Two-component, high-precision, detergent-compatible protein assay. Compared to most dye-binding methods, the BCA assay is affected much less by protein compositional differences, providing greater concentration accuracy. The Dilution-Free BSA Protein Standards, included in the kits, eliminate the need for serial dilutions. |
| Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit | Colorimetric | This kit maintains the key characteristics of the traditional BCA assay but eliminates sample and standard curve dilution steps. Additionally, it only requires a 5-minute, room temperature incubation, similar to dye-binding methods. |
| Qubit Protein Broad Range (BR) Assay Kit | Fluorescence | This fluorescent protein assay provides accurate protein quantification across a broad range (0.1 – 20 mg/mL), and is specifically designed for the Qubit 4 Fluorometer. It is compatible with both reducing agents and detergents. |
| Qubit 4 Fluorometer | Fluorometer | The Qubit 4 Fluorometer is the latest version of the popular Qubit fluorometer designed to accurately measure protein quantity. |
Tips
- No one reagent is the ideal or best method for all applications. 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.
Find assays with Protein Assay Selection Guide Download Protein Assay Technical Handbook
Recommended assays based on common lysis buffers
| Lysis/Extraction buffer | Recommended protein assay | If adding reducing agents or metal chelators (e.g., DTT above 1 mM, EDTA above 10mM) |
|---|---|---|
| RIPA | BCA Protein Assay | BCA Protein Assay-Reducing Agent Compatible |
| M-PER Mammalian Protein Extraction Reagent | BCA Protein Assay | Bradford Assay |
| B-PER Bacterial Protein Extraction Reagents | BCA Protein Assay | BCA Protein Assay-Reducing Agent Compatible |
| NE-PER Nuclear and Cytoplasmic Extraction Reagents | BCA Protein Assay | Bradford Assay |
| Cell Extraction Buffer | BCA Protein Assay - Reducing Agent Compatible |
Tips
- BCA Protein Assays have a unique advantage over Bradford assays, as they are compatible with samples that contain up to 5% surfactants (detergents) and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy.
Recommended assay based on required sensitivity
For quantitation of low volume or very dilute samples, fluorescent assays can provide working ranges down to 10 ng/ml compared to enhanced colorimetric assays at 500 ng/mL and 2,000 ng/ml for standard colorimetric protocols.
| Sample type requirements | Recommended protein assay |
|---|---|
| Very dilute or small sample volumes (minimum sample volume: 1 µL) | CBQCA Protein Quantitation Kit |
Tips
- The CBQCA Protein Quantitation Kit is a very sensitive assay for quantitating proteins in solution, capable of detecting as low as 10 ng of protein per mL. Similar in sensitivity to our NanoOrange protein quantitation reagent (N-6666), CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions, or detergents, and for lipoproteins and small peptides.
Recommended equipment
Once the total protein assay is complete, results can be read using either a UV-Vis spectrophotometer for colorimetric based assays or a fluorometer for fluorescence-based assays.
Multiskan Sky Microplate Spectrophotometer
The Multiskan Sky Microplate Spectrophotometer is a UV/Vis microplate spectrophotometer designed to be convenient and easy to use for virtually any photometric research application, especially DNA, RNA, and protein analysis. It is ideal for multi-user environments where a variety of endpoint, kinetic, and spectral assays are performed. Multiskan Sky instruments are available in three different configurations. Touch screen models offer the flexibility to use the stand-alone instrument or in conjunction with Thermo Scientific SkanIt PC software. The Multiskan Sky model operated solely via SkanIt software is ideal for users who rely on a PC for all operations. Cuvette reading capability is also offered in some models.
Protein quantitation products:
References
- Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
- Gornall, A. G. (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766.
- Lowry, O. H., et al. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
- Smith, P. K., et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.
- Stoscheck, C. M. (1987) Protein assay sensitive at nanogram levels. Anal Biochem 160:301–305.
Detect and measure your target protein
There are various methods that can be used to detect and measure your target protein depending on your experimental needs. Below are common techniques used to detect and measure proteins from complex mixtures (e.g., lysates, sera) and the typical requirements for each.
| ELISA | Western Blotting | Mass Spec | |
|---|---|---|---|
| Advantages |
|
|
|
| Sensitivity | <5–10 pg/mL | Low femtogram to high attogram* | Attomolar range (1018) |
| Lysis buffer compatibility |
|
|
|
| Typical total protein required | 0.1–1 µg/mL | 1–50 µg | <1 µg |
| Equipment required | Plate reader | X-ray film or CCD imaging equipment | Mass spectrometer |
| *With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate | |||
Recommended equipment
ELISA measurement and analysis
Multiskan Sky Microplate Spectrophotometer
In addition to reliable ELISA measurements, perform UV-Vis photometric research applications such as DNA, RNA, and protein analysis with the Thermo Scientific Multiskan Sky Microplate Spectrophotometer. The Multiskan Sky reader features a broad wavelength range (200–1000 nm) path length correction and a fast reading speed. Its intuitive touchscreen user interface, on-board software, and built-in protocols let you run quick measurements directly from the instrument. Alternatively, with any instrument purchase you can use our unlimited license for our easy-to-use Thermo Scientific SkanIt Software, with access to our extensive online library of ready-made protocols.
Western blot documentation and analysis
iBright Imaging Systems
The iBright 1500 Imaging Systems are powerful and easy-to-use, providing sensitive, streamlined, multimode image capture for gel and western blot documentation. The iBright FL1500 Imaging System is capable of easily capturing 4-plex images. It features a large capacitive touch-screen interface and intelligently designed software.
Protein detection products:
| Product/workflow highlights | Category | Description |
|---|---|---|
| Protein gels | Electrophoresis and Western Blotting | Unique protein gel chemistries for all application needs |
| iWestern workflow | Electrophoresis and Western Blotting | An intelligent, start to finish western blotting solution |
| Protein mass spectrometry workflow | Mass spectrometry | Mass spec reagents, instrumentation, and software solutions for proteomics workflows for discovery and targeted proteomics applications |
| ELISA Kits | ELISA | 5 simple steps to achieve consistent and accurate results when running ELISAs |
Tips
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Resources
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Support
For Research Use Only. Not for use in diagnostic procedures.