firstdim

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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	First Dimension Check:


	[check IEF]


	Trouble shooting 1^st dim:


	[Blue Curtain] [horizonal striking]

First Dimension


	The use of GE’s IPGphor together with the “Manifold” is recommended

To achieve a good protein solubilisation in the first dimension, Urea, Ampholytes and detergents were added to the immobilized pH-gradient strips by rehydration.

The normally suggested IPG-strip rehydration-cocktail:
8 M Urea, 0.5% (w/v) Pharmalytes (also included in GE’s IPG-strip buffer) + additives.

....can cause a high background in the second dimension due to unsoluble SDS-Pharmalytes complexes on the
alkaline side of the gel. Use the additional washing steps described for 2D-gels in the different staining methods.
See the 2D trouble shooting “Blue Curtain”.
*Bromophenolblue **Bromophenolred (dyes), ***Bishydroxyethyldisulfide (Aldrich #38,047-4, for pH-gradients including basic region)
* if available

April 28, 2023


	Suggested IPG-strip Rehydration-Cocktail for acidic-neutral pH ranges, f.e. pH 4-7): 10 ml ---> prepare freshly!

8 M Urea	0.75 % (v/v) Ampholytes	0.5% CHAPS	0.28% DTT	*amph. dye “LN”	0.005% BPB*
4.8 g	75 ul (out of 40% w/v)	50 mg	28 mg	50 ul	50 ul (out of 1%)


	Suggested IPG-strip Rehydration-Cocktail for pH-ranges incl. basic pH-values, f.e. pH 4-10: 10 ml ---> prepare freshly!

8 M Urea	0.5 % (v/v) Ampholytes	10% Glycerol	0.5% CHAPS	100 mM BisHED ***	*amph. dye “LN”	0.005% BPB*
4.8 g	50 ul (out of 40% w/v)	1.2 ml	50 mg	100 mg	50 ul	50 ul (out of 1%)


	The Rehydration-Procedure:

11 cm strip	24 cm strip
200 ul	450 ul
6 h min, or overnight	6 h min, or overnight


	Pipet the volume into the lanes of a rehydration-tray (GE is selling them inside a rehydration tray, and place the strips gel-side downward onto the liquids. Wait for 30 min, then cover the rehydration tray with a mineral oil.


	Suggested 2D Sample Solution: 5 ml ---> prepare freshly!

9 M Urea	2 % (v/v) Ampholytes	4 % CHAPS	1 % DTT	*amph. dye “LM”	0.005% BPR**
2.7 g	100 ul (out of 40% w/v)	200 mg	50 mg	25 ul	25 ul (out of 1%)


	Test-sample: E.coli [Sigma #EC-11303]): 1 ml, prepare freshly!

E.coli	2D Sample Solution
30 mg	1 ml


	for basic pH-gradients: 0.5% DTT


	Ultrasonic treatment (5 min) or freeze and thaw 2 times. Centrifuge (5 min à 11.000 rpm).


	Test-sample load: (per lane in a cup at the anode (the acidic end)




		Equilibrator (side 1) Rehydrator (side 2)



	Shell’s “Universal Oil” works nicely!

IPG-Strip	Coomassie	Fluorescencence	Prelabeling	Silver Staining
24 cm strip	35 ul sample + 115 ul rehy.cocktai ~0.5 mg	15 ul sample + 135 ul rehy.cocktail ~200 ug	6 ul sample + 94 ul rehy.cocktail ~60 ug	10 ul sample + 140 ul rehy.cocktail ~150 ug
11 cm strip	15 ul sample + 85 ul rehy.cocktail ~300 ug	7 ul sample + 93 ul rehy.cocktail ~100 ug	3 ul sample + 97 ul rehy.cocktail ~30 ug	5 ul sample + 95 ul rehy.cocktail ~80 ug

The run: (on IPGPhor and the Manifold)
Place the strips into the lanes of the Manifold, gel-side upward, using 2 forceps. Acidic end of the strips to the anode and centered in the middle of the lanes!
Apply the wetted (150 ul bidest.water) IPG-electrode strips (GE 80-6499-14) half-side on the ends of the strips. Apply the cup loading system(s). Press the cups tight with the special tool.
Then load all the 100 ml oil onto the strips (even when only 1 strip is running!) in the Manifold. The sample cups should stay empty after the oil-filling!
Then pipet the samples into the cups and finally add 1 drop oil onto each sample-cup. Start the run.


	GE: IPGphor

			Our current 1D procedure-sequence: 1. Lay the strips film-side down into the lanes of the Manifold 2. Set the sample-cups on the manifold and press them tight with the special cup-pressing bar 3. Fill in the oil in the Manifold’s lanes and control the cups. Important: They should not let the oil in! 4. Pipet the samples in the cups


	First Dimension Check:


	[check IEF]


	24 cm IPG-strip, cup-loading, total time: 16 h (start 5 pm - stop 9 am)




		GE’s IPG-Phor

Volts	uA/strip	µA-start/strip	time	temp (degrees Cels.)
150	(80)	~ 25 uA	3 h	20
300	(80)	~ 50 uA	3 h	20
300 --->1000	(80)	~ 30 uA	6 h	20
1000 --->10.000	(80)	~ 20 uA	1 h	20
10.000	(80)	~ 15 uA	3 h	20


	(only for cup loading), rehydration loading: 5h

	S 40750 Vh, 16 h


	---> = Volt gradient (active)

11 cm IPG-strip, cup-loading, total time: 7 h (start 9 am - stop 4 pm)

		Total time: 7 h (If run stops in the night: Resharpening of the bands: 2 h 10.000 V in the morning)

Volts	uA/strip	uA-start/strip	time	temp (degrees Cels.)
150	(70)	~ 20 uA	1 h	20
300	(70)	~ 30 uA	1 h	20
300 ---> 1000	(70)	~ 80 uA	1.5 h	20
1000 ---> 6000	(70)	~ 80 uA	2 h	20
6000	(70)	~ 80 uA	2.5 h	20


	---> = linear Volt-gradient (active)


	S ~21000 Vh, 6.5 h

MultiPhor II, rehydration loading, normal power supply (3500 V)

11cm IPG-strip, total time: 12 h (start 5 pm - stop 9 am next day: prolongue step 1 to 8h)

Volts	mA	uA-start/strip	W	time	temp (degrees Cels.)
150	2	~25 uA	5	3 h (8h)	20
300	2	~50 uA	5	1 h	20
300 ---> 3500	2	~30 uA	5	3 h	20
3500	2	~20 uA	5	5 h	20

Still also in use:
MultiPhor II + power supply

S ~24.000 Vh, 12 h / 17 h


	---> = linear Volt-gradient (active)


	24cm IPG-strip, total time: 29 h (start 10 am - stop 3 pm next day)

Volts	mA	uA-start/strip	W	time	temp (degrees Cels.)
150	2	~ 25 uA	5	1 h	20
500	2	~ 50 uA	5	3 h	20
500 --->3500	2	~ 30 uA	5	5 h	20
3500	2	~ 20 uA	5	20 h	20


	S ~81.000 Vh, 25 h


	---> = linear Volt-gradient (active)


	EDC Electrophoresis Development & Consulting, Vor dem Kreuzberg 17, 72070 Tübingen (Germany)