Fluorescent Western Blotting: High Sensitivity Detection of Multiple Targets.

Western blotting with fluorescence detection offers the possibility of detecting multiple targets simultaneously on a single blot. Primary antibodies are increasingly available from multiple hosts, and there are now a wide variety of dye labels to exploit multiple imaging channels. If primary and secondary antibodies are selected so that individual targets can be discriminated, multiple antigens can be detected and quantified in a single experiment. Current fluorescence imaging instrumentation offers multiple detection channels and gives sensitivity comparable to other methods. The method described in this article allows multiple targets to be quantified simultaneously and reduces the need for stripping and re-probing. It also allows loading controls to be detected alongside the targets of interest. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Five-plex western blot detection, including tubulin detection for loading control.

Quantitation of protein in samples prepared for 2-D electrophoresis.

The concentration of protein in a sample prepared for two dimensional (2-D) electrophoretic analysis is usually determined by protein assay. Reasons for this include the following. (1) Protein quantitation ensures that the amount of protein to be separated is appropriate for the gel size and visualization method. (2) Protein quantitation facilitates comparison among similar samples, as image-based analysis is simplified when equivalent quantities of proteins have been loaded on the gels to be compared. (3) Quantitation is necessary in cases where the protein sample is labeled with dye before separation (1,2). The labeling chemistry is affected by the dye to protein ratio so it is essential to know the protein concentration before setting up the labeling reaction.A primary consideration with quantitating protein in samples prepared for 2-D electrophoresis is interference by nonprotein substances that may be present in the sample. These samples generally contain chaotropic solubilizing agents, detergents, reductants, buffers or carrier ampholytes, all of which potentially interfere with protein quantitation. The most commonly used protein assays in proteomics research are colorimetric assays in which the presence of protein causes a color change that can be measured spectrophotometrically (3). All protein assays utilize standards, a dilution series of a known concentration of a known protein, to create a standard curve. Two methods will be considered that circumvent some of the problems associated with interfering substances and are well suited for samples prepared for 2-D electrophoresis. The first method (4.1.1) relies on a color change that occurs upon binding of a dye to protein and the second (4.1.2) relies on binding and reduction of cupric ion (Cu2+) ion to cuprous ion (Cu+) by proteins.

Removal of interfering substances in samples prepared for two-dimensional (2-D) electrophoresis.

Biological samples may contain contaminants that interfere with analysis by two-dimensional (2-D) electrophoresis. Lysates or biological fluids are complex mixtures that contain a wide variety of nonprotein substances in addition to the proteins to be analyzed. These substances often interfere with the resolution of the electrophoretic separation or the visualization of the result. Macromolecules (e.g., polysaccharides and DNA) can interfere with electrophoretic separation by clogging gel pores. Small ionic molecules can impair isoelectric focusing (IEF) separation by rendering the sample too conductive. Other substances (e.g., phenolics and lipids) can bind to proteins, influencing their electrophoretic properties or solubility. In many cases, measures to remove interfering substances can result in significantly clearer 2-D patterns with more visible spots and better resolution. It should be borne in mind, however, that analysis of samples by 2-D electrophoresis is usually most successful and informative when performed with minimally processed samples, so it is important that any steps taken to remove interfering substance be appropriate to the sample and only performed when necessary. Procedures for the removal of interfering substances therefore represent a compromise between removing nonprotein contaminants, and minimizing interference with the integrity and relative abundances of the sample proteins. This chapter presents a number of illustrative examples of optimized sample preparation methods in which specific interfering substances are removed by a variety of different strategies.

In-gel peptide IEF sample preparation for LC/MS analysis.

The technique of proteolytically digesting a sample and identifying its protein components by liquid chromatography followed by mass spectrometry (LC-MS) is a widely used analytical tool. Prior fractionation by isoelectric focusing (IEF) may be performed to increase the depth of proteome coverage. Here, we describe a method for in-gel IEF separation of a proteolytic digest that utilizes commercially available immobilized pH gradient (IPG) strips and a widely used IEF instrument.

2-D Western blotting for evaluation of antibodies developed for detection of host cell protein.

Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting.We present here a method using commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2-D electrophoresis procedure gives highly reproducible spot patterns and entire procedure can be completed in less than 2 days. Software analysis enables the straightforward generation of percent coverage values for the antibody when used to probe HCP-containing samples.

Mass spectrometry-compatible silver staining.

INTRODUCTIONThe ammoniacal silver staining method is one of the most sensitive methods used to detect proteins on an SDS-PAGE gel. However, this and other standard silver staining methods are not compatible with mass spectrometry (MS), which is fast becoming the best way to identify proteins isolated on 2D gels. Because the proteins in gels to be analyzed by mass spectroscopy cannot be modified, many of the common sensitizing agents (e.g., glutaraldehyde and strong oxidizing agents) cannot be used. This method is compatible with MALDI and ESI-MS, and it shows an increased ability to deal with semipreparative protein loads without negative staining as compared with other silver staining methods. However, this process is less sensitive than standard silver staining methods.

Staining membrane-bound proteins with coomassie blue r250.

INTRODUCTIONCoomassie Blue R250 permanently stains membrane-bound proteins and is compatible with PVDF and nitrocellulose membranes, but it is incompatible with nylon membranes. This technique is relatively insensitive, with a detection limit of ~1.5 µg of protein. One drawback of Coomassie Blue staining is that it produces a high background that can make interpretation of results difficult.

Staining membrane-bound proteins with ponceau s.

INTRODUCTIONBecause Ponceau S is relatively insensitive (~1 µg of protein), only the most abundant proteins will be visible. However, it is a reversible stain that can be removed completely with H(2)O prior to processing the blots. After staining, a soft lead pencil can be used to record the presence of visible proteins and molecular-weight markers, which will help when aligning the proteins detected on the membrane by western analysis with those in a total protein-stained gel or membrane. Ponceau S is compatible with both nitrocellulose and PVDF membranes.

Staining membrane-bound proteins with colloidal gold.

INTRODUCTIONColloidal Gold is the most sensitive staining technique for proteins bound on membranes, detecting as little as 1-3 ng of protein. Protein spots are permanently stained a dark red after incubation with the Colloidal Gold solution. Colloidal Gold staining can detect proteins on both nitrocellulose and PVDF membranes, but it is not recommended for nylon membranes.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: SDS-PAGE of Proteins.

INTRODUCTIONFollowing first-dimension IEF and equilibration of the IPG gel strips, the proteins are separated on the basis of their molecular weight in the second dimension on an SDS-PAGE gel. Systems for this separation are available from a variety of suppliers and are commonly found in many protein chemistry laboratories. This protocol describes a method for placement of the IPG strip and gives some recommended electrophoresis conditions for these second-dimension gels.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins.

INTRODUCTIONThis protocol describes a method for rehydration of IPG gel strips in preparation for their use for isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. Following rehydration, IEF can be performed using either a flatbed unit or a self-contained instrument.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Isoelectric Focusing of Proteins in a Multipurpose Flatbed Electrophoresis Unit.

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. This method serves as the first dimension of the 2D separation. The method described in this protocol utilizes a flatbed unit; however, self-contained instruments for IEF are also available.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IEF of Proteins in an IEF-Dedicated Electrophoresis Unit.

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels, providing the first dimension of the 2D separation. In this protocol, the IPG gels are focused using self-contained instruments for IEF. These high-voltage systems allow fewer manipulations of the IPG gels, resulting in less error, strip mix-up, contamination, air contact, or urea crystallization. Because rehydration and IEF can be performed consecutively within a single unit, these two steps can be performed unattended overnight. Finally, faster separations and sharper focusing are possible due to the higher voltage available in these instruments.

Phosphoprotein Staining with the GelCode Phosphoprotein Staining Kit.

INTRODUCTIONThe phosphorylation state of a protein has an important role in the regulation of a wide variety of cellular processes. As a result, there has been a great deal of interest in detecting phosphorylated proteins. The method presented here uses the GelCode phosphoprotein staining kit (Pierce Chemical Company). This method depends on the hydrolysis of the phosphoprotein phosphoester linkage using sodium hydroxide in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-molybdate complex is stained with Methyl Green. After destaining, the phosphoproteins are colored green to green-blue. The detection limit is in the nanogram range, but depends on the degree of phosphorylation of the protein. This method will detect the phosphoproteins phosvitin and ß-casein in the 40-80 ng/band and 80-160 ng/band range, respectively. The method presented here is for staining minigels. Volumes will need to be increased for larger gels.

Preparation of Vertical SDS Slab Gels: Casting a Single Homogeneous Gel.

INTRODUCTIONFollowing the separation of proteins by IEF, the second dimension is carried out by SDS-PAGE. This protocol details the method for casting single homogeneous SDS-PAGE gels. Homogeneous gels (with the same %T and %C throughout) offer the best resolution for a particular molecular-weight range and are commonly used because they are the easiest to pour reproducibly. The second-dimension gels can be conveniently prepared in three different formats (i.e., sizes): minigels, for use with 7-cm IEF first-dimension gels; standard gels, for use with 11-, 13-, and 18-cm IEF first-dimension gels; and large-format gels, for use with 18- and 24-cm IEF first-dimension gels. All of the gels use a common set of reagents, listed below, but differ slightly in the equipment required.

Preparation of Vertical SDS Slab Gels: Simultaneous Casting of Multiple-Gradient Gels.

INTRODUCTIONGradient SDS-PAGE gels provide the best resolution over a wide range of molecular weights, resulting in sharper protein spots, because diffusion is minimized by the decreasing pore size in the gel. However, gradient gels are more difficult to produce reproducibly; thus, they are commonly cast with multiple-gel casters, which allows for an identical set of gels to be produced for an experiment. Presented here is a method for casting gradient gels using a multiple-gel casting system.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IPG Strip Equilibration.

INTRODUCTIONThe equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis. Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension. The reductant (dithiothreitol) ensures that disulfide bridges are broken. SDS ensures that the proteins are denatured and also provides a net negative charge to all proteins. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking and other artifacts in the second-dimension separation. Iodoacetamide also alkylates residual dithiothreitol, preventing point streaking and other silver staining artifacts. Finally, a tracing dye (bromophenol blue) is added to allow the electrophoresis to be monitored during the run.