High Performance Gel Electrophoresis (HPGE)

Frequently Asked Questions (FAQ)

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Index: General Operation Zone Elution: Selective Retrieval Concentrator In-Gel Assays Gel Casting Preparation of Samples Labeling Preparation of Concentrators and Retrievers Software Pulse-Field and Temperature Gradients Maintenance Year 2000 Compliance   (began Other Common Questions

• Q: How can I insert gel and buffers?
A: To insert a gel, you open the lid and insert the anodic (the reservoir on the right side with the round indentation) and cathodic (the left reservoir) buffer tank filled with buffer into their positions left and right from the mirrored gel stage first. Press the tanks down firmly on their connectors. Now you can insert the gel on the stage. Hold the geltray in a slight angle and sit it on its back position first, watching for the two Platinum sensor electrodes, which will pierce into the holes in the tray. Then push it firmly towards the back frame of the stage and gently down, until it snaps into position. Adjust the tray horizontally on the stage to line up with the white marks on the frame. Do not forget to peel off the transparent protection film from the gel before starting your experiment!
• Q: What is the temperature range of the system?
A: The system's range for operation is 0°C - 70°C, selectable in 0.1°C intervals. Please keep in mind, that the system controls the temperature in the gel, which means, that it will compensate computer-controlled for the Joule heat generated by the wattage in the gel.
• Q: Can I select the wavelength of the excitation lamp?
A: The light source in the HPGE system was designed for maximum intensity for excitation over a wide UV range. Its energy is evenly distributed from 240-380 nm (maximum at 302 nm, two low energy minima at 254 nm and 280 nm) to allow for broad excitation of a variety of dyes without photobleaching or inducing of nick translations. Thus, you cannot specify a certain wavelength for excitation.
• Q: Can I select the emission wavelength?
A: Yes. The HPGE 1000S system provides capability to selectively visualize only emissions at defined wavelength in 5 nm step within the visible range. You also can select several 5 nm windows simultaneously for discriminative imaging, using multi-color labeling.
• Q: Can the instrument see UV absorbance?
A: Generally, the HPGE detection system is a fluorescence emission detection system. It cannot visualize UV absorbance e.g. to see unlabeled proteins or nucleic acids. Howver, in certain applications the system utilizes intrinsic fluorescence of gel additves to visualize migrating molecules via fluorescence quenching (protein shadowing). In these cases, the detection system can be used to monitor absorbance, although with significant lower sensitivity.
• Q: Which gels can I run on the HPGE system?
A: There are no matrix restrictions in the HPGE sytem. You can run agarose gels, acrylamide gels, copolymers, etc. within a wide variety of gel concentrations, native and with additives (except for organic solvents). Using the 3 mm thick format in gel trays, due to the built-in lane separators it is also possible to run different gel concentrations side by side in a single experiment or to use semi-solid media for very large particles.
• Q: Why do I need to use more dilute buffers than in conventional electrophoresis?
A: The HPGE system utilizes higher field strength to minimize run time and increase resolution. Since most separations are performed in 3 mm thick gels and a larger gel volume than usual, more Joule heat is generated. In order to maximize field strength at reasonable wattage and to guarantee fast and precise temperature control within the gel itself, most protocols use more dilute buffers than conventional separations, typically by a factor of 5 - 10.
• Q: When should I use 3 mm gels, and when 1.5 mm thick gels?
A: If you are interested in semi-preparative or preparative experiments, in cases, where sample concentrations or salt content of samples vary strongly, you need 3 mm gels. You also need 3 mm thick gels for all applications with concentration, selective retrieval (electroelution) or in-gel assays to provide a sufficient entry surface for good electrical contact with the vertical applicator electrode during these processes. For high resolution analytical applications 1.5 mm gels are recommended, which allow higher field strength.

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• Q: Can I elute from any position on the gel?
A: With the exception of the sample slot around 5.0 mm, you can elute at any position of the gel within the range from 0 - 120 mm with a postion accuracy of 0.1 mm.
• Q: Which gels can I elute from?
A: Selective Retrieval works from both, Agarose and Acrylamide gels in geltrays. You need to use 3 mm thick gels for preparative applications. Elution will take longer for larger molecules and from more restrictive gels (e.g. Acrylamide). Typical elution times are in the range of 30 sec. - 5 min. in Agarose, and 2 - 20 min. in Acrylamide.
• Q: Why do I need a gel plug in the retriver, even if I want to elute into solution?
• A: In order to prevent leakage of the elution buffer through the nozzle of the retriever onto the gel surface and to provide a smooth and even contact interface, you need to seal the opening by pipetting 50-100 µl agarose plug gel into the retriever.
• Q: How can I avoid, that my protein is "over-eluted" and lost on the anodic electrode?
A: The HPGE system provides information about the time required at a certain field strength to do a quantitative elution. This information is based on the migration rate (mobility) of the particular zone and allows to predict the distance the zone will move upwards during elution.

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• Q: What can I do with the Concentrator?
A: The Concentrator provides an automated tool for enrichment of minor components in dilute samples. This concentration can be performed in fashion of conventional stacking or in combination with size exclusion chromatography in presence of the electric field for selective enrichment of a certain size range of molecules within a complex mixture. The Concentrator allows sample loads up to 20 ml / lane and concentration down to 5 µl (4000-fold). In combination with ion exchange material, it also can be used for electro-desalting of high salt samples prior to separation.
• Q: What sample volumes can I use?
A: While the standard sample applicator can be used with volumes between 10 - 25 µl (or up to 100 µl using a stacking gel plug), the Concentrator is used for volumes between 3 ml - 20 ml of dilute samples. You should not use the Concentrator with volumes smaller than 3 ml or with salt concentrations in the original sample exceeding 100 mmol.
• Q: Can I use the Concentrator to desalt my sample?
A: Yes. You can perform electrochromatography on ion exchange material in the Concentrator. You have to seal the bottom nozzle with a non-restrictive plug gel, then fill in your ion exchanger.
• Q: Why do I have to take the Concentrator out of the unit before scanning?
A: In every scan, the instrument scans the entire gel surface by moving across the total path of each gel. Since the Concentrator holds considerable amount of material (gel, buffer, sample), it will extends over the normal end position when the carriage moves towards the anodic stop. In order to avoid movement of the carriage with the Concentrator attached, the software will automatically prompt you to take the Concentrator out of the unit when the concentration phase is completed. The system will hold zones stationary until this is done, and will then proceed with scanning.

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• Q: Is it possible to perform enzymatic tests in the gel?
A: Yes. The HPGE system's Assay module allows for enzyme-substrate reactions directly in the gel, either as end point or kinetics. You can use any enzymatic assay, as long as you have a fluorescent substrate, and you can incubate within the entire temperature range. It is also possible to continue separation after completion of the assay.
• Q: Can I use the In-Gel Assay for immuno precipitation?
A: Yes. The Assay module allows for immunological activity determination via fluorescence immuno assays as well as for immuno precipitation detection via zone shadowing.
• Q: How can I generate reaction kinetics curves from peak data?
A: Reaction kinetics in in-gel assays are expressed by increase in peak area over time, detected by periodic interval scans over the area of reagents introduction. For each reaction, you can generate direct activity plots via peak report / time charts for each zone of interest. This way, you can determine several activities at once (e.g from enzyme isoforms or different antigens
• Q: Can I perform multiple assays in one gel at different positions?
A: Yes. Since the HPGE system controls position and extend of reagents within the gel, it is capable of performing multiple asays within a gel. You also can combine immunological and enzymatic assays at different positions.

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• Q: Can I heat up the assembled gel casting device for agarose gelcasting?
A: The gel casting device consists of a sandwich of acrylic plates with a geltray in between squeezed onto a gasket and an anodized Aluminum back plate for even temperature distribution during casting. You may warm up the assembly in a thermostaed forced-air oven or incubator up to 50° C for better flow of high concentration agarose gels. NEVER try to microwave the assemly! It will damage the device and your microwave.
• Q: Do I need the Casting Extender for casting of acrylamide gels?
A: When casting acrylamide gels, it is necessary to avoid air contact on the angled surfaces to ensure proper polymerisation at the buffer interfaces. Thus, it is necessary to fill more acrylamide than required into the leg portion of the gel trays to allow for liqid overlay. This is, what the Casting Extender is for.
• Q: How can I avoid airbubbles around the sample slot formers when filling?
A: There are several ways, depending which type of gels you cast. In any case it is mandatory, that the casting device is clean and dry and its gasket was rinsed thoroughly in distilled water prior to drying. When preparing agarose gels, fill the lanes, so that the slot former are on the upper end of the casting device and the device is slighty angled. In this case, airbubbles will escape towards the top of the tray. Do not fill agarose too hot! Best casting temperatures are between 70°C -65°C. When casting acrylamide gels, make sure, that the mixture was degased for 15 min. prior to addition of the polymerisation starter. Fill quickly horizontally in even stream and allow airbubbles to escape through the extender gasket by gently pushing against the gasket..
• Q: How do I prevent, that the gel breaks, when I take it out?
A: If you prepare Agarose gels, let them sit at room temperature for at least 15 min., then put them in the refrigerator at 4°C for a minimum of 20-40 min. (depending on the Agarose type and the gel concentration). Open the casting device evenly and remove the cover plate from one side slowly under a slight angle while watching the slot former position carefully. Special caution is needed when casting briddle gels like MetaPhor Agarose or high %T, highly crosslinked Acrylamide gels. These gels separate best when adjusted to room temperature.

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• Q: Do I always need to label my samples?
A: No. Although the HPGE system is a fluorescence detection system, there are several ways to find out, how unlabeled samples separate during electrophoresis. You can run a reference standard in an adjacent lane and use it as migration reference, e.g. for elution. Also, in isoelectric focusing the intrinsic fluorescence of carrier ampholytes can be used to detect zones via protein shadowing ("negative staining": dark zones on bright gel background). Lastly, there are moleclues with intrinsic fluorescence, which are always directly visible.
• Q: Which dyes can I use for reversive labeling?
A: There are several reversibly binding dyes available depending on the target molecule. These dyes do not covalently bind to the target. Depending on the nature of the binding, they can be removed by changes in pH or ionic strength.
• Q: Do I need to remove excessive labeling reagents prior to electrophoresis?
A: Typically, you do not need to remove excessive dye, since it will move away from your sample zones quickly. This is the case, because dyes usually are small, highly charged molecules, which are barely restricted by the gel matrix. However, in cases, in which you separate very small molecules (dye and sample components are of comparable sizes), you should consider either using a dye moving in opposite direction than the labeled component, or to minimize the excess of dye during labeling.
• Q: How does labeling influence the mobility of my sample components?
A: The dye you attach during labeling is typically a highly charged molecule, thus it will contribute to the overall charge of your sample. In environments, where separation is primarily a size separation (e.g. SDS separations), this does barely influence the mobility. In charge separations (e.g. IEF), your mobilities will be increased (and your isoelectric point will change). Therefore, for IEF you should not label your sample, but use shadowing in the intrinsic fluorescence of the carrier ampholytes instead.
• Q: What salt concentration can the system tolerate?
A: This depends primarly on the sensitivity of your samples (towards salt and temperature) and the speed you want to run. Since increased ionic strength means increased conductivity, you will generate more heat (higher current) at equivalent V/cm. This means, your runs will typically last longer to avoid too much Joule heat. Because of this considerations, we recommend to use ionic strength < 100 mM.
• Q: How can I clean up samples prior to separation?
A: The best way for on-line sample cleanup is the use of the Concentrator.
• Q: Which are the best fluorescent labels for proteins?
A: The most commonly used dyes for proteins are FLUOS and FITC, which provide quick and versatile labeling. Cascade Blue, Lucifer Yellow and SYPRO Orange are also used. For bi-functional labeling (-SH and amino group), MHS or MBS can be used, but the procedure requires significant sample preparation time..
• Q: Which are the best fluorescent labels for nucleic acids?
A: The most commonly used intercalating dyes are Ethidiumbromide and Ethidium-Homodimer (dsDNA). SYBR Green I can be used with higher sensitivity and slower off-rate. DAPI is a non-toxic, but less sensitive alternative. For 5'-labeling, Fluoresceine is used. The dyes used in automated sequencing instruments can be used, but with much lower sensitivity due to UV-only excitation..
• Q: Which are the best fluorescent labels for carbohydrates?
A: Carbohydrates and Dextrans are best labeled with 2-Aminobenzidine or ANTS.
• Q: Which Polystyrene size markers can I use?
A: Fluorescent Polystyrene microspheres are excellent size markers for very large molecules. Their spherical shape and defined size allows to accurately calibrate very high molecular weights, and they can be used as models for virus separations. The best to use are carboxylated (highly charged) PS Brilliant Blue (468 nm), PS Yellow-Green (540 nm) and PS Yellow-orange (590 nm). PS Polychrome Red (641, 657 nm) is less sensitive.
• Q: Do I have to treat standards and samples the same way?
A: In order to achieve accurate calibrations, you should treat standards and samples as identical as possible. This rule does not apply, if you have pre-labeled ready-to-use standards, but in this case you should check your sample labeling by periodically labeling of reference proteins within the same batch to ensure proper reaction.
• Q: How can I be sure that my quantification is correct, since it is based on fluorescent labeling?
A: To ensure correct quantification with an unknown labeling system, you have to establish a calibration curve. This can be done by serial dilutions of the dye at constant ratio of protein or by dilution series of proteins with a constant amount of dye. Once you have established the range (typically 2-3 orders of magnitude in concentration), you can do exact quantification. As long as your dye is in slight excess (and, since the excessive dye moves off during electrophoresis) and you are within the range of your calibration curve, you will achieve accurate quantification. Note, that the HPGE system also allows for autocalibration in cases of dye-loss (e.g with loosely bound dyes) over time, based on the scan data history.

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• Q: How can I fill the concentrator evenly and prevent leaking?
A: The most important thing is to make sure, that the small amount of plug gel is pipetted into the nozzles the same way you would do to prepare a gel plug for a retriever. After gelation of these plugs, pipett equal amounts of concentrating medium (gel or chromatography medium) in each concentrator chamber while positioning the concentrator in its stand to ensure a proper horizontal filling. The upper buffer shuld be added last after complete gelation or sedimentation of the concentrating medium.
• Q: How do I pipet the Agarose for gel plugs in the retriever?
A: To seal the retriever nozzle opening evenly, make sure, that your Agarose solution is at least 75°C hot before pipetting into the retriever. Set the pipette tip on the ridge inside each nozzle chamber and move it evenly from one side to the other while dispensing the Agarose. This will ensure an even coating and sealing of the nozzle opening.
• Q: Do I have to fill all lanes with upper buffer when using the retriever?
A: No. You should fill only those lanes you want to elute from with buffer,but you have to fill always lane #1 for internal reference reasons. Therefore, if you plan to use the retriever, it is advisable to use lane #1 as a standard lane.
• Q: Why is it not advisable to use Polyacrylamide gel plugs?
A: Since oxigen is a polymerization inhibitor, Polyacrylamide gels will not polymerize evenly when exposed to air. This is particularly true for the small amounts of gel (~50µl per lane) which are used to seal the retreiver nozzle. The result will be rough gel plugs with a high likelyhood for leakage. Also, keep in mind, that Acrylamide gels have narrower pore sizes than Agarose gels, which will result in longer elution times.

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• Q: How can I create a new user?
A: To create a new user, you have to have system administrator's priviledges. You can add or delete users from the menu Configure - Users. Once the user is created, you have to define its level and profile via Configure - Priviledges. This menu item also is used to establish relationship between users for validation.
• Q: Why I am blocked out from certain functions, and my colleague is not?
A: The HPGE software defines three different user levels. Depending on your level, your priviledges vary, so some users may validate (e.g. supervisors), may be able to modify or create methods while others are not allowed to do so. Please contact your system administrator for more details.
• Q: How may scans can I perform in a single run?
A: 60.
• Q: How can I view the event log?
A: Each experiment records automatically events (instrument conditions) and scan data. The event record can be accessed by checkmarking its box when you load the data file. All parameters are provided in a spreadsheet-format window, from which you can select, copy or export to other applications.
• Q: What is an IDB, and what are the limitations?
A: The abbreviation IDB stands for Instrument Data Block. Every IDB contains a set of instrument parameters at a particular time of an experiment and information about the type of event recorded.
• Q: How can I create presentations from my data?
A: The HPGE software provides image capturing and export via cut-copy-paste across the entire Windows platform (e.g. peak data or event data into spreadsheet, graphics packages, word processors..) with direct menu options, which allow quick access to publication-quality output.
• Q: Explain the DataExport function more detailed!
A: HPGE data are stored internally in a proprietory binary data base format. They contain the entire experiment raw data, events, user and validation information. These data can be exported in different formats to make them accessible to other programs for further processing.
• Q: For which calibrations can I use CurveFit, and what are the coefficients for?
A: CurveFit is a program which allows to calculate calibration curves for size/molecular weight determinations, quantitation, isoelectric point calculations, activity measurements or other x/y-dependencies via least-square approximation of up to 3rd order. The resulting coefficients from the standard fit are used in the Calibrate-window to define the calibration function for calculation.
• Q: Which diagnostic functions can I access?
A: Although most of the diagnostic functions are restricted to authorized persons only, you can access some functions directly. The most useful diagnostic function is the test of the UV lamp current, which allows to monitor warmup and scanning behaviour of the excitation light source. You also can perform a series of tests via the diagnostic upgrade feature.

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• Q: What is the difference of pulse field and field inversion?
A: Pulse field is generally a technique, which uses a switched field for separation. This field can be established by a multiple of electrodes at different positions around the gel or just refers to a periodic switched ("pulsed") field. Field inversion is a special case of pulse field, in which the direction of the field from a set of electrodes is inverted in periodic intervals.
• Q: Can I use asymmetric pulsing and field-stop techniques?
A: Yes. The HPGE system allows for asymmetric pulses (time and/or filed strength in forward and backward direction are different) and field-stop techniques (during backward pulse time, no field strength is applied).
• Q: Why is my stage temperature changing frequenly during pulsing and does this effect my separation or gel temperature?
A: The HPGE system regulates the temperature in the gel and automatically compensates for Joule heat generation due to the electric field applied on the gel. Since in most pulse field applications the field strength varies in forward and backward direction, the instrument's temperature controller compensates for the different wattage during each pulse, thus you will see the stage temperature changing. This does not effect your temperature in the gel and therefore has no influence on the separation.
• Q: Why can't I use constant current mode in pulse field?
A: Because pulse field refers to a filed strength gradient in forward-backward direction and a net forward movement of your zones is desired, the current in the gel will change between forward and backward direction. Therefore you cannot use constant current mode together with pulse field.
• Q: Do I need special gels for pulse field applications?
A: In Agarose gel applications not all Agarose types are suited for pulse field separations. You need to use an Agarose which is certified for that pupose.
• Q: How can I use the temperature control setting for TTGE applications?
A: In Timed Temperature Gradient Electrophoresis (TTGE), a temperature gradient is applied over the time of separation.The HPGE system allows for ramping and holding of temperatures by changing the gel temperature either from the status window or from the menu selection Operate - Instrument parameters - Temperature.

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• Q: What areas of the instrument need periodic maintenance?
A: You need to wipe out the interior surface under the buffer wells periodically with a soft sponge with distilled water. Check the calibration targets for liquid spilling. If you notice spots on the targets, wipe them off carefully with a soft, water soaked cloth (Do not wipe them with paper towels, since most contain fluorescent optical whiteners!). Clean the airfilters every 6 month.
• Q: Can I change the excitation lamp?
A: Yes. The user can change the excitation lamp, since it is a self-aligning, self-centering light source. Please observe the directions for lamp change carefully. NEVER turn the instrument on without a lamp connected, and NEVER change a warm lamp!
• Q: Where are the airfilters located, and when should I clean them?
A: The airfilters are located on the bottom side of the instrument. In order to clean them, please take buffer tanks, gel and applicator out of the instrument before you attempt to clean the airfilters. Tilt the instrument sideways, so that the legs are on the left side when you look at the instrument from the front. The main squared airfilter (area under the stage) snaps in and out and should be cleaned like a air conditioner filter. Do not forget to also clean the 7 circular, small airfilters which are secured with a O-ring.

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• Reminder:
 
• Q: Is the HPGE system (hardware and software) Year 2000-compliant?
A: Yes (all Windows'95 versions from 2.12 or higher, using the update kit). The Year 2000 problem in computers ("Y2K-bug") is based on the fact, that computer chips, operating systems and application software mostly relied on only 2-digit date fields, which will turn to "00" at the end of the millenium. Within the HPGE system itself, the embedded code relies on time/date information sent from its application software. LabIntelligence has designed a "Year 2000 Update" software, which addresses several issues (some of which are related to computer manufacturer and operating system manufacturer) in one package, which is distributed free of charge to all registered users..
• Q: What does the "Year 2000 Update" consist of?
A: The update consists of a disk with documentation and self-installing files. It addresses the following issues: 1. Check for computer BIOS compatibilty; if not, apply patch for bootup operation; 2. Check and update of operating system: update of DOS and Windows'95 date/time stamp handling with autheticated patch from Microsoft; 3. Version check and update of the HPGE file system for 4digit year and new index system (all previous data wiil remain compatble and can be converted to the new file system at time of loading, a process coomon to users after some version updates); 4. A log of the successful update will be created. Please read the dcoumentation carefully, which comes with update!..
• Q: I have not received my update disk. When will I receive it?
A: The update disk started shipment end of November 1998. All registered HPGE users will receive notification on their shipment via e-mail. If you are an international customer, please expect delays due to Christmas holiday shipping. You should certainly receive your free update by end of the year.!..

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• Q: How can I increase the sensitivity for visualization in IEF of unlabeled proteins?
A: In IEF, the visualization of proteins depends on shadowing (flourescence quenching) of the intrinsic fluorescence of the carrier ampholytes. In order to increase sensistivity, you have to increase either this background fluorescence or improve the contrast between zone and background.
• Q: What are advantages and disadvantages of black gel trays over clear ones?
A: Black gel trays are used for separations with extreme opaque matrices. These matrices tend to diffuse fluorescent zones when detected in reflectance mode using the stage mirror enhancement.When used with clear trays, this effect makes zones appear broader. To counteract reflection diffusion, the black trays cut any signals which are not directed towards the top of the gel. Thus, signal:noise ratio is improved, although about 1/3 of the emitted light will be lost, which results in a lower sensitivity.

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The HPGE 1000 System from LabIntelligence is a fully automated, computer-controlled real-time gel electrophoresis system for the life sciences. LabIntelligence is located in Belmont, California, U.S.A. For more information, call (+1) 650-654-9908 !

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