FAQ
Definition of %T and %C
The composition of any polyacrylamide gel is given by two parameters:
%T = concentration of total acrylamide
%T = [g (acryl + bis) / 100 ml] x 100
%C = % of the total acrylamide that is crosslinker
%C = [g (bis) / g (acryl + bis)] x 100
The total percent of acrylamide (%T) in the separating gel, which can range from 5 to 20%, determines the pore size. The percent of crosslinker (%C) used is generally 2.6% for all gels. The stacking gel has a very low total acrylamide concentration because the largest possible pore size is required. A typical stacking gel is 4% T, 2.6% C.
Should I run my IEF gel under native or denaturing conditions?
IEF can be run in either a native or a denaturing mode. Native IEF is the more convenient option, as precast native IEF gels are available in a variety of pH gradient ranges. This method is also preferred when native protein is required, as when activity staining is to be employed. The use of native IEF, however, is often limited by the fact that many proteins are not soluble at low ionic strength or have low solubility close to their isoelectric point. In these cases, denaturing IEF is employed.
Running procedures
1. The three options of reswelling solutions given in Table 1 should only be regarded as typical examples that will give good results for most applications. However, whenever required these recipes can easily be modified for further optimization.
Table 1.
| Alt. 1 | Alt. 2 | Alt. 3 | |
|---|---|---|---|
| Application areas: | Water soluble proteins | Proteins with reduced solubility | Proteins with low solubility, e.g. Membrane proteins Lipoproteins |
| Composition: | |||
| Distilled water | 20.0 ml | 19.5 ml | 12.0 ml |
| Pharmalyte 3-10 | 0.5 ml | 0.5 ml | |
| Urea | 9.6 g | ||
| Triton X-100 | 0.1 ml | ||
| DTT | 60 mg | ||
| Total volume | 20.0 ml | 20.0 ml | 20.0 ml |
| Rehydration time: | 1 - 2 h | 1 - 2 h | 15 - 18 h |
Note: All chemicals should be of the highest purity. Double-distilled water should be used.
The presence of carrier ampholytes, a Pharmalyte, not only increases protein solubility but also their electrophoretic migration velocity resulting in shorter focusing times.
Mercaptoethanol (2%) or dithiothreitol (15–50 mmol/l) can be added to avoid oxidation of sensitive proteins.
Glycerol (20–25%) improves solubility of hydrophobic proteins and reduces the risk for urea crystallization.
Lateral band spreading can be reduced by adding acetic acid (2 mmol/l) and applying the sample at the anodic side or adding Tris (2 mmol/l) and applying the sample at the cathodic side.
Triton X-100 can be replaced with other non-ionic or zwitterionic detergents, e.g. CHAPS. Other carrier ampholytes than Pharmalyte 3–10 may also be used.
Alt. 3 in table 1 corresponds to what is used in the first dimension focusing in 2-D electrophoresis. This alternative can be regarded as a standard choice for focusing under denaturing conditions and will normally give high quality results with all kind of samples.
Opening the package
Note 1: Wear clean gloves to avoid contamination of the gel surface, particularly when using silver stain.
Note 2: The gel is packed so that it is faced down to the aluminium foil backing of the package, and the gel support is uppermost.
Note 3: If only half of the gel is to be used, cut the package in half with sharp scissors, reseal the portion to be saved with tape, and store it at –20°C. Remember to identify the polarity of the remaining part.
Open the gel package from the transparent side. Use scissors to cut around all four sides of the package, taking care not to cut either the gel or its transparent backing film.
To simplify gel handling later on, identify the polarity of the pH gradient. The support film has a precut corner which indicates the anodic side of the pH gradient.
Rehydrating Immobiline DryPlate
1. To prevent the gel from adhering to the glass plate fitted with the U-frame, coat the plate with Repel-Silane.
2. Mark the cathodic side of the gel.
3. Wet a clean thick glass plate with a few drops of water and place the gel on the glass plate with the gel side up.
4. Roll the gel with a clean rubber roller to remove all air bubbles from between the glass plate and the support film.
5. Mount the gel in the cassette taking particular care that the U-frame gasket seals also over the cut-off corner of the supporting plastic foil and that the clamps are mounted correctly to avoid leakage.
6. Fill the cassette with the desired rehydrating solution.
7. Leave the gel to rehydrate for the recommended time.
8. Open the cassette and check the gel surface. Remove excess liquid by placing a filter paper moistened in distilled water on top of the gel followed by a dry filter paper on top.
9. Blot the gel by gently rolling the rubber roller under slight pressure over the dry filter paper. Finally remove the filter papers carefully from the gel.
(Since gels rehydrated in detergent containing solutions have less tendency to stick to dry filter paper, they can be dried with a simpler procedure: Place a piece of dry tissue paper (e.g. Kleenex) on the gel, press gently to ensure contact between tissue and gel, and remove the paper carefully)
Preparing the experiment
1. Setting the cooling temperature
Connect Multiphor II electrophoresis unit to thermostatic circulator (e.g. MultiTemp III) and set the desired running temperature. A running temperature of 10 °C is often used except for gels containing urea, which are preferably run at somewhat higher temperatures (15 °C or more) to avoid precipitation of the urea.
Switch on the thermostatic circulator 15 minutes before starting the run.
2. Positioning the gel on the cooling plate
Pipette a few milliliters of insulating fluid (kerosene or light paraffin oil) on the cooling plate of Multiphor II. Position the gel on the cooling plate so that the polarity of the gel corresponds with the polarity marked on the cooling plate. Ensure that no air bubbles are trapped between the gel and the cooling plate.
Electrode strips are used to ensure good electrical contact between the gel and the electrodes. This prevents sparking and allows salt ions from the gel to migrate into the electrode strips where they will stay and not interfere with the separation.
Soak the electrode strips evenly with approximately 3 ml distilled water and remove the excess by using a filter paper. The electrode strips should appear very dry before they are applied to the gel.
Lay the electrode strips along the long edges of the gel. Cut off the parts of the strips which protrude beyond the short ends of the gels using a pair of sharp scissors.
Sample application
There are basically three different methods for sample application. Which method to choose is determined primarily by the sample volume.
1. For 5–20 μl sample volumes: Apply sample directly on the gel, using Immobiline applicator strip. This applicator strip makes it possible to use a multiple syringe which allows quick and simple sample loading, especially when working with large numbers of samples. Check that the contact between the gel and the applicator strip is uniform. Leave the applicator strip on the gel during focusing.
2. For 15–20 μl (and larger) sample volumes: Use sample application pieces. Apply the dry pieces to the gel surface at the desired position(s) in the gradient. Using a micropipette, apply 15–20 μl volumes of sample solution on each piece. To apply larger volumes, use 2 or 3 pieces stacked or laid end-to end. If you want to apply smaller volumes with by this method, trim the paper proportionally before applying it to the gel. Remove the pieces of paper after completing half the total focusing time.
3. For 2–10 μl sample volumes: Apply the sample as droplets directly on the gel surface.
Running conditions
Place the electrode holder on the Multiphor II electrophoresis unit and align the electrodes with the center of the electrode strips. Finally, connect the two electrodes to the base unit and place the safety lid in position. Connect the electrode leads to the power supply. Typical running conditions are listed in Table 2.
Table 2.
| pH range | Voltage (V) | Current (mA) | Power (W) | Volt-hours (kVh) | Time (h) |
|---|---|---|---|---|---|
| 4 - 7 | 3 500 | 5.0 | 15.0 | 7 - 15 | 2 - 4 |
| 4.2 - 4.9 | 3 500 | 5.0 | 15.0 | 15 - 25 | 4 - 7 |
| 4.5 - 5.4 | 3 500 | 5.0 | 15.0 | 15 - 25 | 4 - 7 |
| 5.0 - 6.0 | 3 500 | 5.0 | 15.0 | 15 - 25 | 4 - 7 |
| 5.6 - 6.6 | 3 500 | 5.0 | 15.0 | 15 - 25 | 4 - 7 |
– Decrease the power and current settings proportionally if only part of the plate is being used.
– The settings above are only to be regarded as guidelines. Some proteins focus very slowly and may require as much as 50–60 KVh to give optimal sharp bands. This must be determined experimentally in each case: Run the sample for different times.
– Since there is no gradient drift in the Immobiline DryPlate there is no limitation in the electrophoresis system as such as to how long the experiment can be continued. The limits are set only by the risk of drying out the gel, oxidizing or denaturing the sample.
These risks can be minimized by placing a plastic foil on top of the gel, running at low temperatures, flushing the unit with inert gas (N2) and/or including a reducing agent (DTT or b-mercapto-ethanol) in the rehydration mixture. The surface can also be protected with DryStrip Cover Fluid using Immobiline DryStrip kit.
Sample preparation
Even if Immobiline DryPlate is exceptionally tolerant towards impure samples, best results are still obtained with samples that are free from precipitates. Should aggregation occur at the application point, this can often be overcome by diluting the sample or changing the sample application position.
Best results are generally obtained when the samples are solubilized in the rehydration solution. If this is not possible, the concentration of salt and buffer ions should still be kept at a minimum and, as a general rule, preferably below 50 mmol/l. Excess buffer and salt ions will cause local overheating due to high local currents, which can result in protein denaturation and/or prolonged running times.
Desalting and buffer exchange can be carried out by dialysis, or, more easily, by gel filtration using a prepacked Sephadex G-25 column (Choose NAP-5 Column, NAP-10 Column or PD-10 Column depending on the sample size.).
Sample concentration
In general, Immobiline gels can take much higher sample loads than corresponding gels with carrier ampholytes. Several factors will determine the optimal sample concentration and volume:
1. pH range
2. Number and relative proportions of the components in the sample
3. Sensitivity of the detection method used.
Guide lines: PhastGel Blue R stain detects proteins down to the μg range. Normally 15–20 μl of sample with a concentration of 0.5–10 mg/ml will give good results. Silver staining has about 20 times higher sensitivity. A suitable load in a narrow pH gradient is normally 2–3 times higher than the load in a pH 4–7 gradient.