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Brandon R. Reed
University of Southern Indiana
4/17/2016
The purpose of this manual is to briefly associate new biochemistry research students, under the supervision of Dr. Collins, with the currently used [type/model] capillary electrophoresis (CE) instrument developed by Agilent Technologies. CE serves as a tremendously powerful tool in biochemical studies, and the capability to utilize CE instruments will serve students well in their future careers. Should you wish to download the manual in its entirety, you may do so here.
Introduction
Electrophoresis History, Concepts, & Theory
Electrophoresis is the method by which charged particles may be separated by mass whilst in an electric field. The separation of these particles is easily described as a migration towards its attractive opposite. Typically this is termed ‘slab’ or gel electrophoresis (GE) and takes place in a horizontal or vertical gel matrix. [1] Capillary electrophoresis takes this simple straight forward method and enhances it through automation, reduction of sample size needed, and higher resolution capability. It is solely because of CE’s high resolution and automation that the scientific community was able to take on and finish the Human Genome Project. Though theoretically possible, without CE this would have been extremely challenging due to the billions of base-pair nucleotides contained in DNA. [2]
The development of this instrumentation occurred over a stretch of years, with a paper by Victor Pretorius in the 1970’s suggesting the technique, and the first peer reviewed paper depicting its accomplishment by Dr. Jorgenson and his team in the 1980’s. The first commercial instrument was put on market by 1989 via Microphoretic Systems, Inc. by inventor Robert G. Brownlee and associates. [2] This group also was the first to add on a UV/VIS setup, along with various other applications, to what was a first generation automated instrument. CE automation was a direct byproduct of the development of inexpensive mass produced capillary tubes for gas chromatography (GC), as well as more sensitive detection methods, which allowed for the development of both CE and high performance liquid chromatography (HPLC). [1] System improvements occurred quickly and by the 1990’s more advanced multi-capillary electrophoretic systems had been developed. These systems now play an essential role in what is now the foundation of the field of genomics. [2] As technology continues to develop, CE currently leads the way in the biotechnology industry as a cornerstone method for the separation of various biological and chemical macromolecules. [1] CE’s high resolution, fast read time, and precise elution capabilities along with the growing pool of new techniques and applications developed in each unique field is what enables this method detailed, repeatable, and reliable provisions in regard to charge mass separation.
The theory behind CE’s development stands shoulder to shoulder with GE, being essentially the same action of electrophoresis, only now performed in a polyimide coated capillary tube. The technique itself is efficient for the separation and analysis of both large and small molecules alike. [4] The basic instrumentation and set-up is depicted in figure 1 for both GE and CE, with one vertical and one horizontal illustration of GE. In figure 2 we see what is essentially a simplified illustration using beakers which shows a box diagram equivalent simplification for the instrument, showing the placement of the cathode, anode, buffer solutions, sample injection, light source, and detectors. Figure 3 then illustrates the inside of the capillary while CE is running, showing the direction of electroosmotic flow (EOF) as well as the appropriate vectors of the charged, and neutral species.
Capillary electrophoresis uses an electric field to influence the migration of ions through the capillary. The force (FE = qE) imparted by the electrical field is proportional to its effective charge, q, and the electric field strength, E. The translational movement of the ion is opposed by a retarding frictional force (Ff = fνep), which is proportional to the velocity of the ion, νep, and the friction coefficient, f. The ion almost instantly reaches a steady state velocity where the accelerating force equals the frictional force.
Electroosmosis or electroosmotic flow (EOF) refers to the movement of the buffer in the capillary under the influence of the electric field. The inner surface of a fused silica capillary is covered with silanol groups (Si-OH), which are ionized to SiO– at pH > 2. The negatively charged surface is counterbalanced by positive ions from the buffer, forming the so-called electric double layer. Under the influence of the electric field, the positive ions in the diffuse part of the double layer migrate towards the cathode; in doing so they entrain the waters of hydration, which results in electroosmotic flow. [4] The equations of electroosmotic flow are identical to those developed for electrophoresis, as both phenomena are complementary. The electroosmotic velocity, υep is defined by the following equation.
The driving force behind CE is this unique EOF which develops along the inner side of the silica capillary. [1] This potential is the overwhelming force driving all the sample through the capillary and into the window for detection. The cathode and anode create the differences in the gradient needed for the separation we see as each species elutes out, with the smallest positive charged mass coming first, followed by increasing positive sized species, neutral species, then large to small negative species. [1] This is the basic principle by which CE works.
Example CE Method & Utilization
There are multiple CE methods, but I have selected just one for an example. Capillary Gel Electrophoresis (CGE) is the method of CE which was responsible for allowing the full sequencing of Homo sapien DNA, as well as the current continued efforts towards genomic sequencing of other species. Genetic model organisms such as the fruit fly and humble nematode are some such examples. [3] CGE is performed in a porous gel polymer matrix, typically a cross-linked polyacrylamide. The ratio of cross-linking to monomer are what effectively create the attained pore size, with more cross-linking, from a cross-linking agent used during make-up, yielding smaller pore sizes. CGE is ideal for use with polynucleotide mixtures such as DNA fragments. [1]
Advantages in CGE versus GE primarily concern the distribution of heat throughout the capillary structure as well as the amount of voltage which is able to be applied. The applicable limit in GE typically being 500V, while CE/CGE can have power supplies, due to the fast cooling ability of the capillary tubes, of up to 25kV. This is an enormous dissimilarity, and the capillaries make all the difference. Peak broadening is typically the problem you would run in to running a GE too fast or with too much applied current, due to “thermally driven convective mixing”. [1] The fast cooling prevents this and allows for the higher voltage which speeds up and also increases the resolution of the overall data.
The procedure for sequencing begins with the isolation and processing of DNA from the nucleus of a cell. Once isolated, specific fluorescent dyes which attach to only one of the four bases found in DNA, are added. Once labeled, the DNA is ran through the CGE instrumentation and the sequence is determined based on the dye color sequence observed in the fragments as they elute out from the column. [1] Multiple DNA fragments can be separated simultaneously using this method along with laser beam light sources with four wavelength detection systems. Detectors are typically charge-coupled devices. [1]
Summation
The future of CE and other separation techniques is advancing quickly. Mass spectroscopy seems to be driving the push in CE and eventually I foresee the HPLC and CE revolution will move over to columns, with high pressure systems and applicable changes being seen in this field. This will most likely be made possible due to advances in new detectors being developed. Technology is continually providing more accurate forms in regards to detectors, light sources, and analytical systems as a whole, meaning that electrophoresis will most likely see even more changes in time to come.
Lab Instrument
Setting up the CE instrument and samples
This manual is a derivative work developed in response to the needs of a previous comparative study between CE and GE (with SDS-PAGE) protein extractions concerning Stemonitis flavogenita mobility.* My hopes are that the development of this manual will serve to help students create reproducible and quality CE results in future studies.
To begin, the protein assay used for testing and depicting the new CE methods and sequences developed are outlined in Table 1. This is identical to the CE assay created in the previous work mentioned above, with the one exception of an additional sample which contained a mixture of each protein. This mixture created a broad shifted peak in the original electropherogram, seen in figure 1 at 11.7 minutes, which is believed to be due to the digestion of the proteins by Pepsin**.
For an overview of instrument settings and reagents needed see Table 2. For pre- and post-condition capillary cleaning sequences see Table 3. The condition sequences in Table 3 occur before and following the selected sample method and sequence, in essence cleaning the capillary for the next sample to be tested. Following table 3 are the procedures and calculations needed for determining each portion.
Table 1
| Protein Assay | ||||
| # | Protein | Sample Concentration (g) | ||
| 1 | Myoglobin | |||
| 2 | Pepsin | |||
| 3 | Peroxidase | |||
| 4 | Pyruvate Decarboxylase | |||
| 5 | Bovine Serum Albumin | |||
Table 2
| Instrument Settings & Reagents | |
| Length | 56.0 cm |
| Internal D | 50.0 µm |
| Run Time | 15.0 min |
| UV detection | 277.0 nm |
| Potential | 25.0 kV |
| Polarity | Positive |
| Temp | 25.0 °C |
| Buffer | 25.0 mM Tris Seperation Buffer |
| Buffer pH | 8.25 |
| Base | 0.1 m NaOH |
| Rinse | dH2O |
Table 3
| Capillary Conditioning Sequences | |
| Pre-condition | 0.1M NaOH, dH2O, and Tris Buffer. |
| Post-condition | 0.1M NaOH, and dH2O |
Reagent & Sample Preparation
Turning On & Initializing the Instrument
Follow the bulleted list below for safely starting and initializing the CE instrument. For reference there is a collection of images (Fiugres 2-6) which should help you through the process. It is important to note that this software is expensive and was created to run on Windows 2000, where subsequent versions and upgrades for new Windows systems would correlate to added expenses for the lab. That being said, DO NOT CONNECT THE COMPUTER TO THE INTERNET OR ANY NETWORK. By not doing so you ensure that no update packages will be received via any software vendors connected to the PC. This is per Dr. Collins request.
Turning on the CE
- Turn on the backup battery
- Turn on the CE instrument
- Turn on the computer
- Click the Instrument “online” icon, this opens up the online portion of the CE software, which is the portion that allows for samples to be run.
- Go to File and select a sequence
CE Results & Analysis:
The CE software from Agilent supports multiple output file formats for exporting data (.txt, .csv, .dif, .xls, etc.) so you should have no problem offloading your data for further analysis off-campus. If you are using a flash drive for data storage the computer may not read the drive. I was able to pull data off using an older SanDisk 2.0GB Cruzer drive, but my USB 3.0 SanDisk 32GB drive was not recognized. For all you beginning students let this be a lesson to keep in mind as you go forward. Dr. Collins keeps a ton of old floppy disks because they are the media utilized by her UV camera set-up she uses to take pictures of GE gels. You never know when you might need an older format or drive for something you inherit in a lab. That being said, Google is your friend, and as an example of the internet’s beauty I bring you the 128GB floppy disk. This disk was created using an SD card which was placed into the disk chassis and aligned with the reader pins.
You never know what type of programs you can find in GitHub or elsewhere, developed by our friends over in the Computer Sciences, which may help you transfer formats or ease the pain of expensive hardware becoming obsolete. Never forget that creativity is not a resource quarantined to the arts, for it’s better to be seen as crazed, obsessed even, than unsuccessful. Don’t allow yourself to accept no for an answer, when so plainly anything is possible.
Foot Notes
* S. flavogenita is a model myxomycete which exhibits a complex multi-stage life cycle. During one of these stages, the aphanoplasmodial stage, the myxomycete undergoes cellular changes to become mobile as a slime mold. During this time the organisms are able to move throughout their environment. The previous work, by Thad Whittington and Robert Monsen, was focused on the process concerning this mobility. In particular, the determination of which specific cytoskeletal proteins were present by protein extraction from the organism. The comparison of CE and GE methods for studying these proteins was a subsequent area of focus.
**Pepsin is a protein which is seen in our own anatomy, as a product of chief cells in the stomach and intestinal lining, and is responsible for the initial degradation of protein structures. The development of reproducible instrument settings which yield quality electropherograms, as well as the determination of Pepsin driven protein degradation, is needed for continued research.
References
- Skoog, Holler, and Crouch; Principles of Instrumental Analysis, 6th Edition, pp 857-862.
- DNA Sequencing by Capillary Electrophoresis, Applied Biosystems Chemistry Guide, 2nd Edition, Applied Biosystems, 2009.
- Nora; Instructions for using Beckman PACE MDQ capillary electrophoresis, 2005, Keating Laboratory, Experimental protocols.
- Introduction to Capillary Electrophoresis-A Handbook, Beckman Coulter
- Reed, B.; An Overview of Capillary Electrophoresis as a Separation Technique in Analytical Chemistry, 2015.
- Larsen, S., Bickham, S., Buchanon, T., Jones, W.;Polyacrylamide Gel Electrophoresis of Corynebacterium diphtheriae: a Possible Epidemiological Aid, J. of Applied Microbiology., 1971; 22(5): 885–890.
- Xu, Y.; Tutorial: Capillary Electrophoresis. The Chemical Educator, Vol. 1, 2, 1996.
- Erdmann, P.; The Human Genome Project,1999.
- Landers, James P.; Handbook of Capillary Electrophoresis. New York: C R C P LLC, 1996.
Additional Online Resources, Links, & Videos
- Agilent Technologies
- Beckman Coulter-Introduction to Capillary Electrophoresis
- WileyOnlineLibrary of Electrophoresis
- ChemWiki PDF on Capillary Electrophoresis
- National Institute of Justice (NIJ) Forensic DNA Training Courses
- capillary electrophoresis fundamentals techniques and applications
Instructional Videos
nurvus lab repository 