Ettan IPGphor 3 Isoelectric Focal Bidirection Electrophoresis
1. 10000 V voltage output, semiconductor temperature (15-31°C) once to go 12 samples.
2. Strip slot alumina ceramic surface hydrophobic coating treatment, thermal effect is 100 times more than plastic materials.
3. A variety of tape slots to choose from: standard tape slots can be one step strip swelling, plus samples and IEF electrophoresis; cup-like strips on universal slots for different lengths (7-24cm) of a dry strip can improve sample volume and pH of the protein separation.
4. Manifold accessories box is like a high-throughput cup fittings, plastic and achieves up to 12 simultaneously isoelectric focusing and subsequent balance, providing high-resolution micro-preparative scale protein analysis.
5. IPG dry strips wide range of options between 7-24cm in five lengths, pH3-11.
Ettan IPGphor 3 isoelectric focusing system rules
IPG strips hydration and electrophoretic
1. Using Immobiline DryStrip hydration disk to IPG strips, add the right amount of hydration liquid strips hydration.
2. The installation Manifold strip slot on the Ettan IPGphor electrophoresis. T-shaped protrusions and depressions on the substrate electrophoresis match.
3. In all the tank evenly pour the right amount (108ml) Immobiline DryStrip covering oil, the IPG strip is transferred to Manifold strip groove, glue side up against the bottom of the tank extremely positive mark.
4. Install the sample cup (if a sample cup): A sample cup mounted in the proper position. Relies on the installation tool, the sample can be easily pushed under the cup, fixed to the bottom of the groove.
5. Deionized water wet filter paper performed electrode, and absorb moisture until nearly complete drying, the filter placed at the end of the IPG strip.
6. The electrode over the top of all filters, rotation of the cam to the outer edge of the slot position Manifold strip under the electrode is installed into place.
7. In the sample cup of the sample, up to 150µL. Check to make sure DryStrip cover oil to completely cover the sample.
8. Close the lid, select the desired operating parameters and begin to run glue.
Temperature of 20°C
Maximum current 0.05mAper IPG strip
300V Step-n-hold 3 hours (the sample)
1000V Gradient 6 hours
10000V Gradient 3 hours
10000V Step-n-hold 3 hours
Note: 24cm strips
Narrow pH ranges strips
+30% alkaline pH strips
The balance of PG strips
1. Each 10ml of equilibration buffer before use was added to 100mg DTT (equivalent to the equilibration buffer I), according to Table 2.3 equilibration buffer I was added and the amount of bromophenol blue solution. Remove the IPG strips that were placed in a glass tube (support film close to the wall, each placed in a glass tube IPG strips), sealed with Parafilm, shake 15 min on a shaker, pour equilibration buffer I.
2. The equilibration buffer was added per 10mL 400mg iodoacetamide (equivalent equilibration buffer II). According to the table below add the right amount of equilibration buffer II and bromophenol blue solution. Seal with Parafilm in the shaker oscillation 15min, drain equilibration buffer II.
Table 2.3 IPG strips balanced salt solution with Scale
Strip length is recommended balanced solution volume (ml) / Article bromophenol blue solution (ul)
7 2.5 - 5 12.5
11 5 - 10 25 - 50
13 5 - 10 25 - 50
3. Wash with deionized water run second IPG strips, the edge strip is placed on filter paper for several minutes to remove excess equilibration buffer.
4. The transfer IPG strips: the IPG gel strips on the glass surface located between the support film strips against which to make a piece of glass, with a thin foot gently to the IPG strips push down on the entire surface of the lower edge of the strip of plastic is completely in contact with the plate. Ensure that the glue between the IPG strip and plate glass and plastic as well as support inter-membrane without bubbles.
5. Optional: Add a molecular weight standard protein (molecular weight standard protein solution with an equal volume of 1% agarose solution was mixed and added to the paper can be loaded onto the IEF get good results at a final concentration of 0.5% agarose will. cohesion, before applying a voltage to prevent the proliferation of standard proteins).
6. The final solution is capped by agarose sealing, sealing with a small amount of liquid (about 1-1.5mL) IPG strips that are completely covered, in this process, do not produce bubbles.