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.

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.

Discover more about cell lysis and fractionation

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.

Discover more about protease and phosphatase inhibition

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.

Download Protein Clean-Up Technical Handbook

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 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.
Pierce Rapid Gold BCA Protein Assay Kit Colorimetric This kit maintains the key characteristics of the traditional BCA assay but allows a fast time and room temperature incubation equal to dye-binding methods.
Quant-iT Protein Assay Fluorescence The assay is highly selective for protein and exhibits very little protein-to-protein variation. The assay is performed at room temperature, and the signal is stable for 3 hours.
Qubit 4 Fluorometer Fluorometer The Qubit 4 Fluorometer is the latest version of the popular Qubit fluorometer designed to accurately measure protein quantity.

Use the interactive Protein Assay Selection Guide

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

Tips

  • BCA Protein Assays have a unique advantage over the Coomassie dye-based assays (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

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

  1. 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.
  2. Gornall AG (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766.
  3. Lowry OH et al. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
  4. Smith PK et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.
  5. Stoscheck CM (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
  • High-throughput capability with 96-well or 384-well plate formats
  • Quantification of target proteins
  • Identification and verification of molecular weight
  • Ability to separate and isolate protein of interest
  • Identification and quantitation of multiple targets from the same sample
  • Detection of post-translational modifications or different isotypes
Sensitivity <5–10 pg/mL Low femtogram to high attogram* Attomolar range (1018)
Lysis buffer compatibility
  • For non-activity based ELISAs: ionic detergent based lysis buffers
  • For activity based ELISAs: non-ionic detergent-based lysis buffers (e.g. NP-40, Triton X-100)
  • For SDS-PAGE (denaturing): RIPA or other lysis buffers with ionic detergents
  • For native-PAGE applications: non-ionic detergents-based lysis buffers (e.g. NP-40, Triton X-100)
  • Detergents and high salts must be removed prior to analysis
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 reader instrument

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 FL1500 Imaging System

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

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.

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.

Discover more about cell lysis and fractionation

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.

Discover more about protease and phosphatase inhibition

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.

Download Protein Clean-Up Technical Handbook

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 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.
Pierce Rapid Gold BCA Protein Assay Kit Colorimetric This kit maintains the key characteristics of the traditional BCA assay but allows a fast time and room temperature incubation equal to dye-binding methods.
Quant-iT Protein Assay Fluorescence The assay is highly selective for protein and exhibits very little protein-to-protein variation. The assay is performed at room temperature, and the signal is stable for 3 hours.
Qubit 4 Fluorometer Fluorometer The Qubit 4 Fluorometer is the latest version of the popular Qubit fluorometer designed to accurately measure protein quantity.

Use the interactive Protein Assay Selection Guide

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

Tips

  • BCA Protein Assays have a unique advantage over the Coomassie dye-based assays (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

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

  1. 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.
  2. Gornall AG (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766.
  3. Lowry OH et al. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
  4. Smith PK et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.
  5. Stoscheck CM (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
  • High-throughput capability with 96-well or 384-well plate formats
  • Quantification of target proteins
  • Identification and verification of molecular weight
  • Ability to separate and isolate protein of interest
  • Identification and quantitation of multiple targets from the same sample
  • Detection of post-translational modifications or different isotypes
Sensitivity <5–10 pg/mL Low femtogram to high attogram* Attomolar range (1018)
Lysis buffer compatibility
  • For non-activity based ELISAs: ionic detergent based lysis buffers
  • For activity based ELISAs: non-ionic detergent-based lysis buffers (e.g. NP-40, Triton X-100)
  • For SDS-PAGE (denaturing): RIPA or other lysis buffers with ionic detergents
  • For native-PAGE applications: non-ionic detergents-based lysis buffers (e.g. NP-40, Triton X-100)
  • Detergents and high salts must be removed prior to analysis
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 reader instrument

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 FL1500 Imaging System

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

Resources

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