Polyacrylamide Gel Electrophoresis.

Cross-linked chains of polyacrylamide can be used as electrically neutral gels to separate double-stranded DNA fragments according to size and single-stranded DNAs according to size and conformation. Polyacrylamide gels have the following three major advantages over agarose gels: (1) Their resolving power is so great that they can separate molecules of DNA whose lengths differ by as little as 0.1% (i.e., 1 bp in 1000 bp). (2) They can accommodate much larger quantities of DNA than agarose gels. Up to 10 µg of DNA can be applied to a single slot (1 cm × 1 mm) of a typical polyacrylamide gel without significant loss of resolution. (3) DNA recovered from polyacrylamide gels is extremely pure and can be used for the most demanding purposes (e.g., microinjection of mouse embryos). However, polyacrylamide gels have the disadvantage of being more difficult to prepare and handle than agarose gels. Methods are presented here for preparing and running nondenaturing polyacrylamide gels and for detection of DNA in these gels by staining.

Detection of Radioactive DNA in Polyacrylamide Gels.

Bands of radioactive DNA separated by polyacrylamide gel electrophoresis may be detected by autoradiography or phosphorimaging. Analytical polyacrylamide gels containing radioactive DNA are usually fixed and dried before autoradiography. However, if bands of radioactive DNA are to be recovered from the gel, the gel should generally not be fixed or dried.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Phos-tag SDS-PAGE.

Phos-tagTM SDS-PAGE is a method that enables electrophoretic separation of proteins based on their phosphorylation status. With Phos-tagTM SDS-PAGE, it is possible to discriminate between different phosphoforms of proteins based on their phosphorylation level and the number of phosphorylated sites, and to determine the stoichiometry of different phosphorylation products. Phos-tagTM SDS-PAGE is useful for analyzing disordered proteins with multiple phosphorylation sites and can be used for any of the downstream applications used in combination with conventional SDS-PAGE, for example, Western blotting and mass-spectrometry. To obtain the best results with Phos-tagTM SDS-PAGE, however, it is often necessary to optimize the gel composition. Depending on the molecular weight and number of phosphoryl groups added to the protein, different gel composition or running conditions should be used. Here, we provide protocols for Mn2+- and Zn2+-Phos-tagTM SDS-PAGE and give examples of how disordered proteins with different characteristics behave in gels with various Phos-tag concentrations.

2D Gel Electrophoresis Analysis of Leishmania Proteomes.

2D gel electrophoresis enables resolution of intact proteins in complex mixtures and is thus useful for comparative proteomic analysis, particularly of posttranslationally modified proteoforms that might not be distinguished by shotgun proteomic analysis of peptides. 2D gel electrophoresis is a multistep procedure that can require sample-specific optimization. We present a comprehensive protocol that is effective for 2D electrophoretic analysis of proteins from Leishmania promastigotes and may also be employed for Leishmania amastigotes and for trypanosomes.

Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein

Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactide-co-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.

Purification of Developing Enamel Matrix Proteins Using Preparative SDS-PAGE.

In this chapter we discuss the potential of preparative SDS-PAGE for use in purifying native developing enamel matrix proteins. We believe that the methodology has the potential to provide the relatively large-scale single-step purification of any enamel protein that can be resolved as a single band during analytical SDS-PAGE. Of course, a single band on analytical SDS-PAGE does not guarantee absolute purity as the band may be comprised of two or more proteins migrating at the same apparent molecular weight on the gel. Where absolute purity is required, the methodology can be used in conjunction with other techniques such as ion-exchange chromatography or reverse-phase chromatography. We do not see preparative SDS-PAGE replacing chromatographic methodologies but believe that it can provide another powerful tool to add to the battery of purification techniques already available to researchers in the field.

A modified clear-native polyacrylamide gel electrophoresis technique to investigate the oligomeric state of MBP-5-HT3A-intracellular domain chimeras.

The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering. Here, we demonstrate the accuracy of this ensemble approach by characterizing the previously established pentameric state of the intracellular domain of serotonin type 3A (5-HT3A) receptors.

Methods for Monitoring Macroautophagy in Pancreatic Cancer Cells.

Macroautophagy is a catabolic process through which redundant, aged, or damaged cellular structures are first enclosed within double-membrane vesicles (called autophagosomes), and thereafter degraded within lysosomes. Macroautophagy provides a primary route for the turnover of macromolecules, membranes and organelles, and as such plays a major role in cell homeostasis. As part of the stress response, autophagy is crucial to determine the cell fate in response to extracellular or intracellular injuries. Autophagy is involved in cancerogenesis and in cancer progression. Here we illustrate the essential methods for monitoring autophagy in pancreatic cancer cells.

Two-Dimensional Gel Electrophoresis by Glass Tube-Based IEF and SDS-PAGE.

The genome information combined with data derived from modern mass spectrometry enables us to determine the identity of a protein once it is isolated from a complex mixture. Two-dimensional gel electrophoresis established more than four decades ago serves as a powerful protocol to isolate many proteins at once for such protein analysis. In the first two decades, the original procedure to use a glass tube-based IEF had been commonly used. Since an IEF in glass tubes is rather difficult to maneuver, a new method to use an IEF on a thin agarose slab backed by a plastic film (IPG Dry Strip) had been invented and is now widely used. In this chapter, we describe a protocol that uses a glass tube-based IEF because the capacity of protein loading and resolving power of this type of classic two-dimensional gel is still indispensable for many applications, not only for protein identification but also for protocols that are benefited by larger amounts of materials, i.e., analysis of posttranslational modification of proteins such as phosphorylation, methylation, glycosylation, and others.

Tris-Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass.

Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-acetate-PAGE system a very helpful tool for protein analysis.

Two-Dimensional Gel Electrophoresis: Vertical Isoelectric Focusing.

Two-dimensional gel electrophoresis is one of the most powerful tools for separating proteins based on their size and charge. Two-dimensional gel electrophoresis (2-DE) is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

A Combined Free-Flow Electrophoresis and DIGE Approach to Compare Proteins in Complex Biological Samples.

Free-flow electrophoresis has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility and wide applicability to protein classes. As a stand-alone platform, however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with free-flow electrophoresis to simultaneously separate and identify differentially expressed proteins in a model cell system.

A Multichannel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High-Throughput Protein Separation and Characterization.

We developed a multichannel gel electrophoresis system that continuously collects fractions as protein bands migrate to the bottom of gel columns. The device uses several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "counter-free-flow" elution technique allows continuous and simultaneous fraction collection from multiple channels at low cost. Using the system with SDS-PAGE, 300 µg samples of protein can be separated and eluted into 48-96 fractions over a mass range of 10-150 kDa in 2.5 h. Each eluted protein can be recovered at 50% efficiency or higher in ~500 µL. The system can also be used for native gel electrophoresis, but protein aggregation limits the loading capacity to about 50 µg per channel and reduces resolution. This system has the potential to be coupled with mass spectrometry to achieve high-throughput protein identification.

Isolation of Proteins from Polyacrylamide Gels.

Minute amounts of proteins are required for immunization of mice for the development of antibodies including monoclonal antibodies. Here we describe a rapid procedure for the isolation of proteins from polyacrylamide gels after sodium dodecyl sulfate polyacrylamide gel electrophoresis in sufficient amounts for immunization of animals.

Continuous Elution SDS-PAGE with a Modified Standard Gel Apparatus to Separate and Isolate an Array of Proteins from Complex Mixtures.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a powerful tool to separate proteins according to their relative sizes. The technique provides information about both the size of a number of proteins and potentially the comparative amounts of each protein. To confirm the identity of proteins, proteins can be eluted from the gel and transferred electrophoretically to nitrocellulose for antibody-based detection. During electrophoresis, if the current is not stopped, proteins continue to pass down the gel and elute from the bottom of the gel. The standard electrophoresis gel apparatus can be employed with the addition of some tubing and alterations to the separating gel to collect proteins separated by size as they elute from the base of the gel as described in this chapter. Complex protein mixtures can be separated into multiple fractions containing single proteins in a few hours. Small amounts (<500 µg of protein) of protein sample can be fractionated.

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100 µg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

Ultrarapid Sodium Dodecyl Sulfate Polyacrylamide Mini-Gel Electrophoresis.

We describe here an ultrafast method for electrophoresing proteins on SDS-PAGE. Previously we reported a method to complete SDS-PAGE and immunoblotting in an hour, including electrophoresing proteins at 70°C in 10 min. Here we show that we can electrophorese molecular weight standards and bovine serum albumin on a 4-20% gradient gel in well under 10 min using heated (44 °C) Laemmli running buffer and high voltage.

A Brief Review of Other Notable Electrophoretic Methods.

Electrophoretic methodologies for qualitative and preparative purposes are commonly used in biological research and have been well established as an integral analytical tool for a long time in most research laboratories. Listed here are some of the more specialized innovations that have been developed in recent times for special purposes of study. These include micropreparative isoelectric focusing in liquid suspension, accelerated protease digestion by SDS-PAGE, two-dimensional SDS-PAGE for membrane protein resolution, carbon nanotube-modified page for resolution of complement C3, electrophoretic resolution of ultra-acidic proteomes in acidic media, and two-dimensional immunoelectrophoresis of pre-beta/alpha lipoprotein A-I in agarose. All these methods are briefly reviewed in this chapter.

Ex-Vivo Signal Transduction Studies in Chronic Lymphocytic Leukemia.

Microenvironmental signaling is pivotal to chronic lymphocytic leukemia (CLL) pathology; therefore understanding how to investigate this pathway by both protein and chemical methods is crucial if we are to investigate and correlate biological changes with therapeutic responses in patients. Herein, we describe the use of western blotting also referred to as immunoblotting as a method that can semiquantitatively evaluate changes in protein expression following receptor engagement; this includes B cell receptor (BCR) signaling following stimulation with anti-IgM (Blunt et al. Clin Cancer Res 23(9):2313-2324, 2017). It is important to note that immunoblotting should always be combined with other quantitative methods such as flow cytometry to confirm activation of these signaling pathways (Aguilar-Hernandez et al. Blood 127(24):3015-3025, 2016).

Application of Gelatin Zymography in Nanotoxicity Research.

Gelatin zymography is a relatively simple, inexpensive, and powerful technique to detect proteolytic enzymes capable of degrading gelatin from various biological sources. It has been used particularly to detect the two members of the matrix metalloproteinase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), due to their potent gelatin-degrading activity. MMP-2 and MMP-9 are also able to degrade a number of extracellular matrix molecules including type IV, V, and XI collagens, laminin, and aggrecan core protein, thus making them important in the nanotoxicity research. In this technique, proteins are separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and gelatinases, activated by SDS, digest gelatin embedded in the gel. After staining with Coomassie Brilliant Blue R-250, areas of degradation are visible as clear bands against a blue-stained background. Here, we describe the detailed procedure for gelatin zymography.