Running a Multiphor II Protocol

Ensure that the electrodes in the Immobiline DryStrip tray are connected and place the lid on the Multiphor II unit. Connect the leads on the lid to the power supply. Ensure that the current check on the EPS 3501 XL Power Supply is switched off. Begin IEF.

As isoelectric focusing proceeds, the bromophenol blue tracking dye migrates toward the anode. Note that the dye front leaves the Immobiline DryStrip gel well before focusing is complete, so clearing of the dye is no indication that the sample is focused. If the dye does not migrate, no current is flowing. If this occurs, check the contact between the electrodes and the electrode strips. Also check that the electrode leads and bridging cable are correctly connected, and check that the electrodes are positioned properly so that the marked side contacts the side rail.

The protocols below are suitable for running 7–24-cm Immobiline DryStrip gels on the Multiphor II Electrophoresis System connected to EPS 3501 XL Power Supply.

The focusing times in the tables below are guidelines only. They may vary with the nature of the sample and how the sample is applied. If using crude samples with high protein and salt content or using paper-bridge loading, the run time in total kilovolt-hours should be increased by 10%.

Guidelines for running Immobiline DryStrip gels on Multiphor II Electrophoresis System. Running conditions: Temperature 20 °C; current 2 mA total; power 5 W total. Program EPS 3501 XL Power Supply in gradient mode and with current check option turned off.

7-cm strips

pH intervals	Step	Voltage (V)	Time (h)	kVh
3–11 NL 3–10 6–11	1 2 3 Total	200 3500 3500	0:01 1:30 0:40–1:05 2:10–2:35	2.8 2.2–3.7 5.0–6.5
3–10 NL 4–7 3–5.6 NL	1 2 3 Total	200 3500 3500	0:01 1:30 0:55–1:30 2:25–3:00	2.8 3.2–5.2 6.0–8.0
7–11 NL	1 2 3 Total	300 3500 3500	0:01 1:30 1:10–2:02 2:40–3:30	2.9 4.1–7.1 7.0–10.0
5.3–6.5 6.2–7.5	1 2 3 Total	300 3500 3500	0:01 1:30 2:36–3:45 4:06–5:15	2.9 9.1–13.1 12.0–16.0

11-cm strips

*To adjust this protocol for an overnight run, extend step 1 by 5 h (2.5 kVh) and reduce step 3 by 2.5 kVh.

pH intervals	Step	Voltage (V)	Time (h)	kVh
3–11 NL 3–10 6–11	1 2 3 Total	300 3500 3500	0:01 1:30 1:45–2:35 3:15–4:05	2.9 6.1–9.1 9.0–12.0
3–10 NL 4–7 3–5.6 NL	1 2 3 Total	300 3500 3500	0:01 1:30 2:20–3:30 3:50–5:00	2.9 8.1–12.1 11.0–15.0
7–11 NL	1 2 3 Total	300 3500 3500	0:01 1:30 3:30–4:55 5:00–6:25	2.9 12.1–17.1 15.0–20.0
5.3–6.5 6.2–7.5	1 2 3 Total	500* 3500 3500	0:01* 1:30 7:10–9:10 8:40–10:40	3.0 25.0–32.0 28.0–35.0

13-cm strips

*To adjust this protocol for an overnight run, extend the time of step 1 to 2h.

pH intervals	Step	Voltage (V)	Time (h)	kVh
3–10 3–11 NL 6–11	1 2 3 Total	300 3500 3500	0:01 1:30 3:10–4:00 4:40–5:30	2.9 11.1–14.1 14.0–17.0
3–10 NL 4–7 3–5.6 NL	1 2 3 Total	300 3500 3500	0:01 1:30 3:45–5:10 5:15–6:40	2.9 13.1–18.1 16.0–21.0
7–11 NL	1 2 3 Total	500 3500 3500	0:01 1:30 5:10–6:20 6:40–7:50	2.9 18.1–22.0 21.0–25.0
5.3–6.5 6.2–7.5	1 2 3 Total	500* 3500 3500	0:01* 1:30 10:00–12:50 11:30–14:20	3.0 35.0–45.0 38.0–48.0

18-cm strips

*This step is added to give a convenient overnight run (15 h). This step may be omitted. Step 4 should then be extended by 2.5 kVh.

pH intervals	Step	Voltage (V)	Time (h)	kVh
3–10 3–11 NL 6–11	1 2 3 Total	300 3500 3500	0:01 1:30 3:10–4:00 4:40–5:30	3.0 17.0–22.0 20.0–25.0
4–7 3–10 NL 3–5.6 NL	1 2 3 Total	300 3500 3500	0:01 1:30 3:45–5:10 5:15–6:40	3.0 3.0 19.0–30.0 25.0–36.0
6–9 7–11 NL	1 2 3 4 Total	500 500 3500 3500	0:01 3:00 1:30 5:25–9:25 12:55–16:55	1.5 3.0 35.5–45.5 40.0–50.0
5.3–6.5 6.2–7.5	1 2 3 Total	500* 3500 3500	0:01 1:30 19:10–23:25 20:40–24:55	3.0 67.0–82.0 70.0–85.0

18-cm strips

*This step is added to give a convenient overnight run (15 h). This step may be omitted. Step 4 should then be extended by 2.5 kVh.

pH intervals	Step	Voltage (V)	Time (h)	kVh
3–11 NL 3–10	1 2* 3 4 Total	500 500* 3500 3500	0:01 5:00* 1:30 8:30–11:20 15:00–17:500	2.5 3.0 29.5–39.5 35.0–45.0
3–10 NL 4–7 3–7 NL 3–5.6 NL	1 2 3 Total	500 3500 3500	0:01 1:30 12:00–16:20 13:30–17:50	3.0 42.0–57.0 45.0–60.0
6–9 7–11 NL	1 2 3 Total	500 3500 3500	0:01 1:30 16:20–22:00 17:50–23:30	3.0 57.0–77.0 60.0–80.0
3.5–4.5	1 2 3 Total	500 3500 3500	0:01 1:30 22:00–27:40 23:30–29:10	3.0 77.0–97.0 80.0–100.0
5.3–6.5 6.2–7.5	1 2 3 Total	500 3500 3500	0:01 1:30 30:35–36:20 32:06–37:50	3.0 107.0–127.0 110.0–130.0