In laboratory environments across the world, gel electrophoresis is a crucial technique that permits the separation of macromolecules such as DNA, RNA, and proteins. Different separation media and mechanisms enable subsets of these molecules to be more effectively separated by exploiting their physical characteristics. For proteins, however, polyacrylamide gel electrophoresis (PAGE) is often the technique of choice.
What is the polyacrylamide gel electrophoresis, and what is protein electrophoresis?
PAGE IN SDS vs. PAGE IN SDS
What is the effect of polyacrylamide gel electrophoresis (PAGE) on the planet?
Protein gels are often described as being ineffective in the system.
What is the electrophoresis of 2D gel?
Protein electrophoresis'' applications
Assay for Electrophoretic mobility shift
- Western blot
Extraction for mass spectrometry
Electrophoresis of Urine protein and immunofixation
In this article, we will discuss how PAGE works, how it can be used, and how it may be interpreted.
What is the electrophoresis of polyacrylamide gel, and what is protein electrophoresis?
PAGE is a method that separates macromolecules based on their electrophoretic mobility, namely, the ability of analytes to move towards an electrode of the opposite charge. This is due to the charge, volume, and duration of the molecule, which is dependent on the protein''s apparent size, and consequently on their ability to move through the pores. It may therefore be desirable to denature proteins prior to electrophoresis to refine them if a more accurate estimate of their dimensions is required.
The pores formed in polyacrylamide are smaller than those of agarose, which is used for electrophoresis in the agarose. This makes it more suitable for the separation of proteins over large polynucleotide DNA or RNA fragments and permits the separation of relatively small proteins. Consequently, when people refer to protein electrophoresis, the separation technique they will most often be referring to is PAGE.
SDS PAGE vs native PAGE
PAGE can be used in denaturing or non-denaturing situations, depending on the purpose of the analysis.
In a series of experiments, an anionic detergent called sodium dodecyl sulphate (SDS) is combined with heat and sometimes a reducing agent is used to denature proteins prior to electrophoretic separation. Proteins are linearized and complex with the SDS, so that all proteins have a similar mass-to-charge ratio. This technique is typically used to separate proteins of 5250 kDa.
Figure 1: SDS PAGE Linearization of proteins. -mercaptoethanol Reduces disulfide bonds, while SDS negates differences in mass-to-charge ratios, allowing proteins to be separated on the basis of molecular weight.
These bonds are maintained in native PAGE, while protecting the proteins'' higher order structure. Consequently, the distribution of proteins through the gel is primarily influenced by the proteins charge (determined by its amino acid sequence and post-translational variations) and the pH of the separation rather than its size in kDa. However, it may be desirable when examining bound proteins or complexes, for example when their biological activity remains intact. Reduced voltages may also be used for separation.
What is the effect of polyacrylamide gel electrophoresis (PAGE) on your body?
PAGE is to divide analytes by passing them through the pores of a polyacrylamide gel using an electric current. This, however, increases the speed at which small proteins are able to move through the gel, thus increasing their resolution and preventing them from going into the buffer rapidly after the current is applied.
Figure 2: Polymerization and crosslinking of acrylamide. APS catalyzed by TEMED leads to polymerization and crosslinking of acrylamide. The total concentration of acrylamide components and the ratio of acrylamide to bisacrylamide have an impact on the gels'' pores and therefore the range of protein sizes that may be resolved.
Regardless of how the PAGE gel is being used, the equipment used is the same. Unless you are switching between using SDS PAGE and native PAGE, make sure that all equipment is properly cleaned and dried before proceeding. Depending on the size, the gel will be evaluated and dried thoroughly before being shipped. Protein-based residues may also affect the gel when stained.
A electrophoresis tank, power pack, and electrophoresis frame are all required to discharge the gel.
PAGE needs three types of buffer:
- Gel casting buffer (used to make the gel)
- Sample buffer
- Running buffer (fills the gel tank where electrophoresis takes place)
A continuous or discontinuous buffer system may be used for protein separations, but it is rarely used for protein analysis. A discontinuous buffer system, most often used for protein separation, uses different buffers for the gel and running buffer. Both layers (stacking and resolving gels) have differing pore sizes and different buffer compositions. The discontinuous buffer system is usually able to produce higher resolution separations.
PAGE is used for tris-based buffers, with tris-glycine, bis-tris, tris-acetate, and tris-tricine, all with added SDS. Tris-borate-ethylenediaminetetraacetic acid (TBE) is the most commonly used for native PAGE. Differences in pH and ionic strength are a result of the discontinuity of the buffering.
3. The gel
The stacking gel and the resolving gel have two sections, each with a higher percentage of polyacrylamide, which is typically higher in the analytes. SDS PAGE is also the preferred treatment method for those who use it.
The resolving gel must be thoroughly cleaned before it starts to set, thus eliminating the need for bubbles. Gels should be placed in an airtight container with a little water to ensure that there are no air bubbles on the surface. The stacking gel should now be placed on the top and the comb is removed, ensuring that there are no chemicals left behind. However, the time period between the two gel layers will take longer to spread. The resultant separation may suffer.
Precast gels are possible, but they are typically less costly than making your own.
4. Probe preparation
The length of time you prepare the sample may vary depending on whether you are performing a SDS PAGE or a native PAGE experiment.
Samples such as lysed cells, tissues, or bacteria are mixed with a loading dye that has several crucial ingredients. Glycerol helps to weigh the sample down and prevent it from falling out of the well during loading. SDS and -mercaptoethanol are often able to linearize the proteins present, but reduce charge differences.
PAGE, SDS, and -mercaptoethanol are not included in the loading dye, and no heating step is performed in order to maintain proteins in their native conformation.
Samples should be centrifuged (16100 x g for 2 mins should be sufficient) to remove insoluble debris. Only the supernatant should be loaded as particulates may alter the flow of the samples down the gel.
Size markers are normally included on the left side of a sample row to determine whether bands are detected. Reference proteins as well as positive and negative controls should be included where possible to verify observations in unknown samples. Where a strain or cell line the same as that of the unknown sample but in which the gene encoding the target protein has been removed might be a suitable negative control. Controls that mimic the unknown samples as closely as possible can be especially useful when looking at samples that aren
6. Running the gel
Part-fill the electrophoresis tank with an appropriate running buffer, often tris-glycine-SDS for SDS PAGE and TBE for native PAGE.
Remove the gel from the casting frame and insert it into the electrophoresis frame. Combine this carefully in the electrophoresis tank and top up with a running buffer to ensure that the wells are submerged. Make sure that samples are cleaned before the comb has been removed, otherwise samples may be cross-contaminated.
Place the lid on the tank, ensure that the electrodes are correctly applied on the surface (black to black and red to red) and determine whether or not an SDS or native analysis is being performed. The voltage used and run time will vary depending on the percentage resolving gel being used, the analyte size, and whether an SDS or native analysis is being implemented.
Figure 3: Sample preparation to protein electrophoresis in PAGE. 1) Samples are prepared for analysis, 2) gels are cast and the equipment is prepared, 3) buffer is added to the gel tank, and samples/controls are added to the gel, 4) current is applied to the samples to separate the proteins, 5) gels are stained and visualized.
7. Staining and visualization
The gel frame is removed from the tank and the gel is carefully removed from the glass plates. The stacking gel has done its job and can be carefully removed and discarded, leaving only the resolving gel.
The proteins must be stained, which is normally achieved with Coomassie brilliant blue, 5 an organic dye that is compatible with the essential amino acids, then staining them in place. So, carefully leave the gels intact, except for the staining technique. Gels must then be rinsed in water and placed in a little water to prevent dehydration.
Solutions that stain proteins without need for destaining are available, which may be particularly helpful if a rapid result is required. However, they are typically more expensive than the standard staining protocol. Other stains, such as silver staining,6 may also be used for specific purposes, but Coomassie staining is the most common.
Interpreting protein gels
Similar to those of agarose DNA gels, images can be taken with a camera to help a long-term picture. Marker ladders are typically run alongside protein samples to allow their length (normally in kDa) to be estimated, but these limitations may cause bands to fall short of strength. These limitations may be imposed on individuals who have chosen a specific sample. In these instances, it is recommended to repeat the analysis with a smaller sample.
Figure 4: A SDS PAGE with Taq DNA Polymerase. A SDS PAGE is a useful technique to separate proteins according to their electrophoretic mobility. The marker (left hand lane) is the Precision Plus Protein Standard all Blue. This SDS PAGE was performed to determine the molecular weight of Taq DNA Polymerase.
What is 2D gel electrophoresis?
Sometimes, separation of proteins in a single dimension is not sufficient to resolve similar species. In situations such as this, separation in two dimensions may increase the required resolving power, as it is less likely that two molecules will be very similar in two distinct properties. Joachim Klose7 and Patrick H. OFarrel.8 introduced a two-dimensional polyacrylamide gel electrophoresis in 1975.
Proteins are then separated linearly according to their isoelectric point (which relates to their charge and pH). In the second dimension, molecules are then separated at 90 to the first separation according to molecular mass to produce an electropherogram (Figure 5).
Applications of protein electrophoresis
PAGE is equited across many biological disciplines, from molecular biology and forensics to biochemistry and genomics, and is now available as an analytical and diagnostic tool. Lets look at some of the methods employed by PAGE as an important component.
PAGE''s samples can be used directly to provide a variety of useful indicators, including:
- if an expected protein is present
- what size (or apparent size) it is
- approximately how much there is
- how pure the protein sample is
- if cleavage (e.g., from a tag) has been successful
- which fraction a protein is present in (e.g., from cell lysate fractions)
- protein solubility
When preparing recombinant proteins,9 it is important to have the ability to verify that the protein of interest has not been lost at multiple stages of the purification process and that it is expected to be full. Depending on the fraction in which it is present, it may also indicate whether there are issues with protein solubility, which may impact its function or binding abilities or preclude purification in the conditions used. Recombinant proteins are essential for vaccine and biopharmaceutical development, as well as diagnostic assays
Protein analyses are also vital for many industries, including food and beverage development,10 quality control,11 safety 12, 13 and fraud detection, and the analysis of environmental samples. 15, 16 However, as technologies are evolving and becoming more affordable and accessible, some analyses in these areas are being replaced17 by techniques such as mass spectrometry (MS).
As part of this process, electrophoretic mobility shift assays (EMSAs) are an important experimental tool in identifying nucleic acidprotein complexes. This can help to identify binding sites such as those used by transcription factors.18 While agarose gels are often used to achieve this as they facilitate the migration of larger DNAprotein complexes, PAGE can help more separation resolution and stability for some complexes.19
Separation of sample proteins is an essential first step to any western blot experiment, and PAGE is often the technique used to accomplish this. While PAGE gels may be stained with Coomassie brilliant blue to serve as loading controls when it comes to interpreting western blot results, however, they may also be used to evaluate infectious and non-infectious diseases, check recombinant protein purification20 and provide functional or validative information to omics investigations.
Proteins in gel bands may be excised and purified for further analysis. MS can be used to obtain sufficient information about proteins found in the sample.
PAGE may be a useful diagnostic tool to detect the quantities of certain proteins in bodily fluids, such as urine or blood. Agarose gel electrophoresis may be used as an alternative for these analyses.
The urine protein electrophoresis (UPEP) test, which involves albumin and globulins, can be used as an indicator of pathological changes. High levels of protein in urine can be an indicator for numerous diseases, including inflammation, kidney disease,21, 22 infection, and some types of cancer (e.g., myeloma)23 and can help to develop further investigation or treatment.
On serum samples, albumin and immunoglobulins24 can be used, as well as in serum samples, to detect multiple myeloma,25 lymphoma, leukemia, kidney disease, liver disease, and malnutritional diseases.
In these tests, immunoglobulin A (IgA) lambda27 may be used to identify certain subtypes of a protein, e.g., the heavy and light chain type of the M protein.28 and 29.
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13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of palytoxin. Nat. Prod. Commun. 2017;12(8):1934578X1701200815. doi:10.1177/1934578X1701200815
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SDS-PAGE technique for fish toxicological experiments. Toxicol. Rep. 2018;5:905-909. doi:10.1016/j.toxrep.2018.08.020
SDS-PAGE heat-shock protein profiles of environmental Aeromonas strains, according to Osman KM, Amin ZMS, Aly MAK, Hassan H, and Soliman WS. J Microbiol. 2011;60(2):149-154. PMID: 21905633
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Proposed and challenges for Recombinant protein expression in Escherichia coli, 20. Rosano GL and Ceccarelli EA. Front Microbiol. 2014;5. doi:10.3389/fmicb.2014.00172
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23. Brandhorst D, Wetter O. SDS-PAGE study of uroproteins in myeloma patients
An underused but very useful study, according to Vavricka SR, Burri E, Beglinger C, Degen L, and Manz M. Serum protein electrophoresis. Digestion. 2009;79(4):203-210. doi:10.1159/000212077
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Proteins: Analysis and Interpretation in serum, urine, and cerebrospinal fluid, according to Clarke W. Marzinke MA. Academic Press; 2020:365-390. doi:10.116/B978-0-12-815499-1.00022-3
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