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Relative densitometry from SDS PAGE

Relative densitometry from SDS PAGE


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I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software.

My questions are about what estimates can be made, given that Coomassie staining can be nonlinear has protein-to-protein variability. Unfortunately, I do not have a standard curve, and the lanes contain total lysate.

  1. Can I take the percentage of band volume to lane volume as an estimate of percentage of total cell protein mass? (25% vs 20% total cell protein)

  2. Or would it more appropriate to take the band volume normalized to an arbitrary band in the lysate, and then report the percentage difference between conditions? (6 vs 4.8 normalized band volume, or 20% decrease in expression levels).


A protocol for recombinant protein quantification by densitometry

Susana María Alonso Villela, TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France.

Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia

Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia

Faculté de Médecine de Tunis, Université Tunis El Manar, Tunis, Tunisia

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

Susana María Alonso Villela, TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France.

Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia

Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia

Faculté de Médecine de Tunis, Université Tunis El Manar, Tunis, Tunisia

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France

TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France


How Does SDS-PAGE Work?

Electrophoresis is a major technique for separating proteins and other substances such as nucleic acids, purines, pyrimidines, some organic compounds and even inorganic ions. Most of the current electrophoresis is to separate the sample into the mobile phase in an immobilized medium. Polyacrylamide gel is one of the main media. It is a porous gel whose pore size is close to the size of protein molecules, which improves the resolution of proteins. Moreover, the polyacrylamide gel has good chemical stability, strong repeatability, stability to changes in pH and temperature, and easy color observation. SDS polyacrylamide gel electrophoresis (SDS-PAGE) has the advantages of simple operation and good reproducibility in the determination of protein molecular weight, detection of specific proteins, and identification of strain species.

Polyacrylamide gel is composed of acrylamide and cross-linking agent N, N'-methylenebisacrylamide under the action of catalysts ammonium persulfate (AP) and N, N, N', N'-Tetramethylethylenediamine (TEMED). It is a gel with a three-dimensional network structure. PAGE can separate proteins into several bands according to the different mobility caused by the different charge and molecular weight of protein molecules. SDS is an anionic surfactant, which can break the hydrogen and hydrophobic bonds of proteins in the presence of reducing agents (β-mercaptoethanol or dithiothreitol, DTT), and combine with protein molecules in a certain ratio to form short rod-shaped composites of the same density. Positively correlated with the molecular weight of the protein, the length of the complex formed by proteins of different molecular weights is different. SDS makes the amount of negatively charged protein far exceed its original charge, masking the natural charge difference between various protein molecules. Therefore, the mobility of various protein-SDS complexes during electrophoresis is no longer affected by the original charge and molecular shape, but only depends on the relative molecular mass.

The polyacrylamide gel is usually composed of a stacking gel in the upper layer and a separating gel in the lower layer. The difference between the upper and lower gels is the concentration of acrylamide and the pH of Tris-HCl. During electrophoresis, an electric field is applied to the gel, and negatively charged proteins migrate across the gel from the negative electrode to the positive electrode. The most common electrophoresis buffer consists of Tris and glycine. The pH in the stacking gel is 6.8, and only a few glycine molecules dissociate. Therefore, the SDS-treated protein molecules move between the upper glycine molecule and the lower Cl- ion. This process compresses the protein sample in the gel into bands that are much smaller than the volume initially loaded. As the electrophoresis progresses, the protein moves to the separating gel (pH 8.8), where of the glycine molecules dissociate. The speed of the movement increases and exceeds the protein. In the separating gel, the speed of movement of each protein depends on its molecular weight. Proteins with small molecular weights can pass through the pores in the gel easily, while those with large molecular weights have more difficulty passing through. After a period of time, proteins reach different distances according to the sizes, achieving the purpose of protein separation.

Figure 1. Schematic diagram of polyacrylamide gel electrophoresis (Gülay, et al, 2018).

How to Determine Molecular Weight of Protein by SDS-PAGE?

SDS-PAGE is the main method to determine the molecular weight of unknown proteins. A protein with known molecular weight and an unknown sample are electrophoresed at the same time. After staining, according to the relative mobility of the standard protein and the logarithm of the molecular weight, a line can be obtained and determine the molecular weight of the unknown sample using its relative mobility. In the laboratory, a standard molecular weight protein covalently coupled to a dye is used as a reference protein to roughly indicate the size of the unknown protein. This pre-stained protein marker can be directly observed during electrophoresis or when transferring membranes.

How to Read SDS-PAGE Results?

After electrophoresis, protein separation cannot be directly observed by the naked eye, and subsequent staining techniques are needed. Coomassie brilliant blue staining and silver staining are common methods for routine detection and quantification of proteins separated by electrophoresis. After simple processing such as fixation-staining-decolorization, the distribution of protein can be clearly observed. With the improvement of high-sensitivity protein analysis methods and protein identification technologies, new staining methods such as fluorescent labeling and isotope labeling technology have greatly improved sensitivity, and are also compatible with automated proteome platform gel cutting technology. More high sensitivity and automated dyeing technologies are been developed.

How to Store SDS-PAGE Gel?

Freshly SDS-PAGE gels are usually prepared before each experiment. However, gels can also be stored in clean water at 4°C for about a week. If the gel cannot be photographed in time after dyeing, it needs to be placed in water to prevent drying and shrinking of the gel. It is advised to photograph the staining results as soon as possible. Band will disperse if the gel is soaked in water for a long time.

References
1. Smith B J. SDS Polyacrylamide Gel Electrophoresis of Proteins. Methods in Molecular Biology, 1984, 1(4):41-55.
2. Duffy M F, Noormohammadi A H, Baseggio N, et al. Polyacrylamide gel-electrophoresis separation of whole-cell proteins. Methods in Molecular Biology, 1998, 104:267.

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Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20-30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE.

Keywords: apparent size gel mobility immunoblotting protein identification protein migration.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

TM-mimetics switch migration positions relative…

TM-mimetics switch migration positions relative to reference proteins on SDS/PAGE at various acrylamide…

Molecular size and net charge…

Molecular size and net charge of TM-mimetics are larger than those of reference…

SDS/PAGE mobility shifts of TM-mimetics…

SDS/PAGE mobility shifts of TM-mimetics relative to reference proteins. ( A and B…


Example of a Standard Curve for Molecular Mass

Protein standards for gels are purified polypeptides with relative mobilities that correspond closely to their true molecular mass. Suppliers of chemicals for electrophoresis such as SIGMA (St. Louis, MO) or Bio-Rad (Hercules, CA) provide ready-made molecular mass standards. SIGMA sells standards for calibrating SDS gels with a tris based buffer system (Laemmli gels).

Inserts with commercial standards, older literature, and even my old web pages may refer to molecular weight (MW) rather than molecular mass. MW is the same as relative molecular mass (Mr) but differs from molecular mass in that it is a unitless quantity. You will likely find MW data that are reported in Daltons or kiloDaltons although such usage is not correct. A number representing MW is identical to the corresponding molecular mass in Daltons, thus the terms can be used interchangeably as long as the units are dropped from quantities that are represented as MW.

SIGMA Standard Mixture for Molecular Weights 30,000-200,000 (SDS6H2)

  • myosin from porcine muscle, 200,000
  • beta-galactosidase, from Escherichia coli, 116,000
  • phosphorylase B, from rabbit muscle 97,400
  • albumin, bovine 66,000
  • albumin, from chicken egg white 45,000
  • carbonic anhydrase from bovine erythrocytes, 29,000

SIGMA Dalton Mark VII-L Standard Mixture, MW range 14,000-70,000 (SDS-7)

  • albumin, bovine 66,000
  • albumin, from chicken egg white 45,000
  • glyceraldehyde-3-phosphate dehydrogenase, from rabbit muscle 36,000
  • carbonic anhydrase from bovine erythrocytes, 29,000
  • trypsinogen, from bovine pancreas 24,000
  • trypsin inhibitor, soybean, 20,100
  • alpha-lactalbumin, bovine milk 14,200

Note that the sources of proteins are varied. You won't find all of them in any one protein fraction, in fact you aren't likely to find any of them, depending on the fraction you are studying. They are used to calibrate gels, not as indicators of what types of proteins are present. Keep in mind that many very different proteins have similar molecular weights. The patterns given by standard mixes become recognizable with experience.

A typical plot of the molecular weights of standards versus their relative mobilities is shown below, using a log scale for the molecular weights. Be very careful with curve fits, keeping in mind that the scale is logarithmic. You don't want to be in error near the top of the gel. It may be better to simply interpolate results (connect the data points).

Relative mobility for a given polypeptide will vary with gel density (%T). Curves will always shift when percent acrylamide changes.

With some gels of high percent acrylamide, the dye front will not be evident, since proteins small enough to run with the same mobility as the bromphenol blue dye may not be present. Unless the position of the dye front was marked before staining, a true relative mobility cannot be determined. As long as a gel is calibrated using internal standards, MW estimates can be obtained by representing relative migration distance using an arbitrary reference point such as the bottom of the gel. Use of a true relative mobility allows one to use the same standard curve for any gel of the exact same composition regardless of dimensions or position of the dye front when electrophoresis is terminated. Error in MW estimates is compounded with the use of an arbitrary reference point when the original dye front was curved or otherwise distorted.


When working with hazardous materials, it is important to be alert to the specific nature of the dangers that are posed by those materials. This also means it is important to identify and differentiate one hazardous material from another in order to effectively apply precautionary measures. Knowing how to read an SDS is a must for managers, executives, and their employees.

Health & Safety professionals know that the ability to understand and read a Safety Data Sheet (SDS) is a fundamental skill for any workplace committed to employee safety.

Although a full 16-section SDS is required by the Globally Harmonized System, not every section addresses the same type of information that an employee or safety manager will need. In this article, I’ll be covering the section related to hazards associated with using/handling materials. By understanding these sections, you’ll be equipped to put the proper preventative and protective measures in place to reduce the risk of injury.

If you’re looking on information about how to read an SDS for emergency response measures you can read this related article “Everything You Need to Know About Safety Data Sheets (SDS) in an Emergency”

The following sections of the GHS Safety Data Sheet provide information on the hazards presented by the material and the specific properties which can be used to identify the material:

  • Section 1. Chemical Product and Supplier’s Identification
  • Section 2. Hazards Identification
  • Section 3. Composition/Information on Ingredients
  • Section 9. Physical and chemical properties

Section 1: Chemical Product and Supplier’s Identification

In this section, general information regarding the material and the supplier are listed. This includes any means of identification for the product (e.g. Product Code, Product Name). These means of identification will give a point of reference between the Label and the SDS, allowing a user to quickly find the proper SDS, should it need to be accessed.

This section also includes information and emergency contact information for the user to get any additional information regarding the product or seek help in the case of an emergency.

Finally, this section will include information regarding recommended uses of the material, or any uses recommended against. This information allows the user to make sure that the material is used in line with the manufacturer’s recommendations.

Section 2: Hazards Identifications

In this section, the risks and hazards associated with the material are identified. This communicates to the user in what ways this material may be dangerous as wells as some general guidelines towards safely using the material. This is also the section in which the elements that need to be displayed on a label can be found.

This section includes the following information:

  • GHS Hazard Classifications
  • Pictograms
  • Signal Word
  • Hazard Statements
  • Precautionary Statements
  • Other Hazards (or Hazards Not Otherwise Classified)

The GHS Hazard classifications are further subdivided into Physical Hazards, Health Hazards and Environmental Hazards. These classifications allow the user to be aware and on the lookout for those hazards. The classifications listed under the GHS Hazard Classifications determine all the information found in the rest of this section, with the exception of Other Hazards (or Hazards Not Otherwise Classified).

The Pictogram, Signal Word, and Hazard Statements give the user a quick reference and description of the hazards. The Pictograms provide a visual representation of the nature of the material’s hazards, the Signal Word communicates severity of the hazards with one word (Warning or Danger), and the Hazard Statements describe the effect of each hazards in one short sentence (e.g. H300 – Fatal if swallowed). This provides a concise and effective description of what the user should look out for when using this material.

The Precautionary Statements can be subdivided in the following categories:

  • General Precautionary Statements
  • Precautionary Statements related to preventative measures
  • Precautionary Statements related to incident response
  • Precautionary Statements related to storage conditions
  • Precautionary Statements related to disposal of the material.

These standardized phrases offer general guideline to mitigate risks, to respond to incidents with preliminary first-aid or fire-fighting advice and to properly store and dispose of the material.

The final category in this section is the Other Hazards. This can also be referred to as Hazards Not Otherwise Classified. This category communicates any additional hazards that are not covered by the GHS, such as risks of dust explosion or biohazards.

Section 3: Composition/Information on Ingredients

In this section, the chemical composition of the material is listed. This includes the chemical name, CAS number and concentration or concentration range of the hazardous chemicals. This allows the user to identify which chemicals are the principal contributors to the GHS Hazard Classifications and implement specific procedures or protective equipment to use to mitigate those hazards.

In addition, the GHS classifications of the pure chemical can be included in Section 3, as well as other means of identification such as EC number or other registration numbers.

Section 9: Physical and chemical properties

In this section, the descriptive physical and chemical properties of the material are provided. This allows the user to identify material in cases of improper secondary container labeling or spills, and it allows the user to make sure the product they are using matches the description given by the supplier. Any significant deviation from this description could indicate something wrong with the material or a different material entirely. Here is a list of commonly listed properties and how that information can be used to increase work place safety.

Density

This is a measure of the mass of material for a given volume. This can also be found as “Relative Density” or “Specific Gravity”, both of which are the density of the material divided by the density of water.

This property will indicate how this material will behave in relation to other materials (e.g. a lower density than water indicates that a material would float on water). This can be very useful in the event of a spill, allowing the user to predict whether or not this material will be on the surface, at an intermediate layer or sinking to the bottom of a mixture based on the densities of all materials involved.

Appearance

This is a visual description of the material, including physical state, color, and consistency. This gives a reference to the user for a visual confirmation of the identity of the material.

This can also help visually identify an unlabeled or spilled material.

Odor description and threshold

This is a description of the expected odor of the material and the vapor concentration at which a person would usually start detecting that smell. This is additional information that allows to identify the product and provides a benchmark to estimate of vapor concentration based on smell.

In other words, by knowing the odor description and threshold, a user can identify whether or not the material is present in the air at dangerous levels (based on Permissible Exposure Levels listed in Section 8).

Water Solubility

This is a description of how well the material mixes with water. Based on the information listed for this physical property, a user can predict the expected behavior of the product if it is mixed with water (e.g. whether it will form a separate phase or dissolve completely).

Flashpoint

This is the temperature at which this product will catch fire when exposed to an ignition source such as a spark or flame. The flashpoint provides more specific information on the flammability of the product and helps identify which products should be stored at colder temperatures to avoid fire hazards.

Freezing/boiling point

These are the temperatures at which the product melts or freezes, and boils. They indicate the temperature at which the physical state of the material would change which helps identify which temperatures might be too low to store the product, since freezing could damage containers and cause leaks, and which temperatures might be too high to store the product, leading to losses through evaporation.

PH

This is a numerical indicator of acidity or alkalinity, based on the pH scale. Acidic products have a pH below 7 and alkaline products have a pH above 7. This physical property helps identify how corrosive product might be and helps assess which other products are incompatible (products with low pH and high pH should be stored separately).

Learning to Read an SDS: Training Required

Because the information listed in the above sections of an SDS (in addition to other sections you’ll find in the 16-section GHS SDS) is important for knowing how to identify, store, and use a chemical product, it’s not only your responsibility to provide your employees with accurate and up-to-date SDSs – you are also required by OSHA, WHMIS, and CLP regulations to provide adequate training to your staff about how to read and understand an SDS.

Training regulations vary according to the Health & Safety regulator overseeing your business, but in general the guidelines require you to:

  • Provide training to employees about the Globally Harmonized System (GHS) standards.
  • How to read all 16 sections of an SDS.
  • Provide access to copies of SDSs for any material to which they have a risk of exposure.
  • To provide SDSs according to federal SDS language laws. You may or may not be required to provide training in a language other than English if you employees cannot adequately understand verbal English (for example, see OSHA OSHA 29 CFR 1910.1200(h)).

Where to Get More Information on SDS Compliance

Want to learn more about what goes in to each section of an SDS and how the information plays a vital role in preventing injuries and mitigating risk? ERA is providing an on-demand free webinar that will give you a deep-dive into SDS sections, SDS authoring, and how it all relates to Health & Safety compliance.


Acknowledgements

The instruments and reagents for CE-SDS and Simple Western were provided by ProteinSimple, a Bio-Techne Brand. We would like to thank Udo Burger, Susanne Doerks and Chris Heger for their tremendous support. We would like to thank the Institute of Pharmaceutical Biology of Technische Universität Braunschweig for the provision of the imager and the working group of Prof. Dr. Ott of the Insitute of Medicinal and Pharmaceutical Chemistry of Technische Universität Braunschweig for lending us the SDS-PAGE system of Bio-Rad. Furthermore, we would like to thank the state Lower Saxony for providing the fellowship for Rebecca Wiesner. For the technical support and advices, we thank Holger Zagst and Matthias Stein. We would like to thank Nordilyn Lorenz for sample preparation and performance of many experiments on SDS-PAGE, CE-SDS and Simple Western.

Open access funding enabled and organized by Projekt DEAL.

Hermann Wätzig likes to mention, that he stands in close and very friendly contact with ProteinSimple for a long time. The authors have declared no other conflict of interest.

Filename Description
elps7314-sup-0001-SuppMat.docx2.2 MB Supporting information file: Comparison of the reference MWs according to manufacturer's specification with the MWs from UniProt Influence of various sample buffers on the MW determination by 10% SDS-PAGE Influence of the denaturation temperature on the MW determination on CE-SDS (n = 3) Influence of the denaturation temperature on the MW determination by CE-SDS and Simple Western (n = 3) Influence of the reducing agents on the MW determination by CE-SDS and Simple Western (n = 3) Electropherograms of the CE-SDS separation of Matuzumab reduced with various reducing agents and non-reduced Matuzumab Comparison of MW markers of five different manufacturers by 10% SDS-PAGE, Electropherograms of the five MW markers on CE-SDS, Linear regression plots based on MW versus the relative migration distance (Rf) respectively the reciprocal relative migration time (RMT) of MW markers using the example of the Precision Plus Protein Standard from Bio-Rad, MW Marker Maurice CE-SDS by ProteinSimple, ProteomeLab™ MW Sizing Standard by Sciex, Unstained Protein Standard by New England Biolabs and the Benchmark™ Unstained Protein Ladder by Novex by life technologies.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


Relative densitometry from SDS PAGE - Biology

Well Volume
1 empty
2 empty
3 5 ul STDs
4 *10 ul Sample A
5 *10 ul Sample B
6 *10 ul Sample C
7 *10 ul Sample D
8 *10 ul Sample E
9 *10 ul AM
10empty

1. Why did you add Laemmli sample buffer to your fish samples?

To linearize the proteins by ensuring the proteins will only move by size, also coating the proteins to be elctriclly charged so they will migrate down the gel.

2. What was the purpose of heating the samples?

To further denature th proteins by reducing disulfide linkages.

3. How are the proteins extracted from the fish samples?

By incubataing the tubes for five minutes at room temperature

4. Have all the proteins been extracted from the fish slice or are some still left after the extraction? How could you test your hypothesis?

Yes, there were none left. This hypothesis could be tested by running a second SDS PAGE.

1. Why do SDS-coated proteins move when placed in an electric field?

SDS gives the protein an overall negative charge with a strength that is relative to the length of its polypeptide chain, allowing the mixture to move according to size, or not at all.

2. What is the purpose of the actin & myosin standards and the Precision Plus Protein Kaleidoscope prestained standard?

The move ahead of the gel. Actin and myosin standards allow comparison of the major conserved muscle proteins. Precision plus protein kaleidoscope standard allows visibility as proteins migrate through the gel.

3. Which proteins will migrate the farthest? Why?

Small negatively charged proteins will migrate farthest because they have less molecular weight.

4. What is the purpose of the stain?

The stain creates the movement of standards and proteins to move along the electrophoresis gel that is visible. This allows the bands to be comparable from the fish samples standards.

1. Which two fish have the most similar protein profiles?

2. Which two fish have the least similar protein profiles?

3. Give an explanation for why you think the protein profiles of some fish species share more bands than other fish species?

These fish evolved differently to their environments than other fish.

4. Did your predictions from your Pre-Lab Activity turn out to be true or not? If not, why do you think that was?

We said catfish and flounder were most closely related due to their swim type and swim environments. This prediction was partially correct because of the matching bands on the graph, however Catfish and Cod are most similar.


Isolation and characterization of photosystem II of Porphyra yezoensis Ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyra yezoensis Ueda (P. yezoensis) by sucrose density gradient ultracentrifugation. After P. yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem II (PSII) particles were isolated and purified. The activity of PSII particles was determined with DCIP (2,6-dichloroindophenol) photoreduction reaction. The composition of purified PSII particles was detected by SDS-PAGE. As a result, seven proteins including 55 kD protein, 47 kD protein, 43 kD protein, 33 kD protein, 31 kD protein, 29 kD protein, and 18 kD protein were found. Compared with PSII particles of higher plants and other algae, they were identified as D1/D2 complex, CP47, CP43, 33 kD protein, D1, D2 and cyt c-550 respectively. Besides, other three new proteins of 20 kD, 16 kD and 14 kD respectively were found. Among these extrinsic proteins, the 16 kD and 14 kD proteins had not been reported previously, and the 20 kD protein was found for the first time in multicellular red algae.


Tricine–SDS-PAGE

Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine–SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine–SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1–2 d.

*Note: In the version of the article initially published online, the words “Gel buffer (3x)” were missing in the table on page 18. The error has been corrected in all versions of the article.


Watch the video: Quantifying bands on SDS-PAGE using ImageJ (February 2023).