Template for proteomics (2D gel)

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MIAPE Gel Electrophoresis Data Entry Worksheet						Double Click buttons to activate them
Worksheet version 1.1 / MIAPE: GE version 1.0 / 2007-09-05						Mouse over field names to view guidance

1			General features
		1.1.1	Date stamp	11-05-2008
		1.1.2	Responsible person or role	(i) Holger Janssen; (ii) University of Rostock, Germany / Institute of Biological Sciences / Division of Microbiology / Albert-Einstein-Str. 3 / 18051 Rostock; (iii) holger.janssen@uni-rostock.de
		1.1.3	Electrophoresis type	two-dimensional

2			Sample	Add another sample	Remove this sample	← Double Click buttons to activate them
		2.1.1	Sample name(s)	Proteome AB2 pH 5.7 C.ac.
		2.1.2	Loading buffer	9.5 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 100 mM DTT, and 0.6 % (v/v) ampholyte (Pharmalyte® pH 3.0–10.0)

3			Gel matrix and electrophoresis protocol	Add another GM/E protocol	Remove this protocol	← Double Click buttons to activate them
	3.1		Dimension details
		3.1.1	Ordinal number for this dimension	first	second
		3.1.2	Separation method employed	IEF	SDS-PAGE
	3.2		Gel matrix
		3.2.1	Description of gel matrix	IPG strip	denaturing gel
		3.2.2	Gel manufacture	-	12.5 % Acrylamide; 0.35 % Bisacrylamid; 25 % 1.5 M Tris-HCl pH 8.8; 0.1 % SDS; 0.025 % APS; 0.05 % TEMED
		3.2.3	Physical dimensions	x = 18 cm; y = 3.3 mm; z = 0.5 mm	x = 25 cm; y = 25 cm
		3.2.4	Physicochemical property range and distribution	pH 4-7	180-10 kDa
		3.2.5	Acrylamide concentration	-	12.15 %
		3.2.6	Acrylamide : Bisacrylamide ratio	-	35 : 1
		3.2.7	Additional substances in gel	-
		3.2.8	Gel lane	-	1
		3.2.9	Sample application	-	1. (reference 2.1.1); 2. (reference 2.1.2); 3. 300 µg protein; 4. (reference 3.2.8)
	3.3		Protocol
		3.3.1	Running buffer	mineral oil (Immobiline DryStrip Cover Fluid)	0.025 M Tris-HCl; Glycine 0,19 M; 0,1 % SDS
		3.3.2	Additional buffers
		3.3.3	Electrophoresis conditions	gradient IEF with 1 mA and 5 W at 20 °C in 4 steps: 1. 2 h with 500 V and 500 Vh; 2. 2 h with 500 V and 1000 Vh; 3. 2 h with 3500 V and 10,000 Vh; 4. 9 h with 3500 V and 31,500 Vh	second dimension: 1500–2500 mW per gel at 12 °C for approx. 18 h

4			Inter-dimension Process — Protocol	Add another IDP protocol	Remove this protocol	← Double Click buttons to activate them
		4.1.1	Step name	equilibration step 1	equilibration step 2
		4.1.2	Inter-dimension buffer	50 mM Tris–HCl [pH 6.8], 6 M urea, 30% [v/v] glycerin, 4% [w/v] SDS	50 mM Tris–HCl [pH 6.8], 6 M urea, 30% [v/v] glycerin, 4% [w/v] SDS
		4.1.3	Additional reagents	3.5 mg/ml DTT	45 mg/ml Iodacetamide
		4.1.4	Equipment	-
		4.1.5	Protocol	strip equilibration for 15 min	strip equilibration for 15 min after equilibration step 1

5			Detection (if applicable)
	5.1		Direct detection
		5.1.1	Name of direct detection process	Coomassie staining procedure (1.5 l Coomassie solution)
		5.1.2	Direct detection agents	1.125 g Coomassie Brilliant Blue G-250 (AppliChem)
		5.1.3	Additional reagents and buffers	112.5 g ammonium sulfate, 375 ml metahnol, 22.5 ml ortho-phosphoric acid
		5.1.4	Equipment	-
		5.1.5	Direct detection protocol	staining at room temperature over night (max. 24h)
	5.2		Indirect detection
		5.2.1	Name of indirect detection process	-
		5.2.2	Transfer medium	-
		5.2.3	Detection medium	-
		5.2.4	Indirect detection agents	-
		5.2.5	Additional reagents and buffers	-
		5.2.6	Equipment	-
		5.2.7	Indirect detection protocol	-

6			Image Acquisition	Add another acquisition	Remove this acquisition	← Double Click buttons to activate them
	6.1		Acquisition Equipment
		6.1.1	Type of equipment	transmitted light scanner
		6.1.2	Name of equipment	UMAX Power Look 2100 XL (Biostep GmbH, Jahnsdorf, Germany)
		6.1.3	Software	Argus X1 (Biostep GmbH, Jahnsdorf, Germany) and Delta 2D (Decodon, Greifswald, Germany)
		6.1.4	Calibration	Yes
		6.1.5	Equipment specific parameters
	6.2		Acquisition Protocol
		6.2.1	Image acquisition process
		6.2.2	Reference to gel matrix

7			Image	Add another image description	Remove this image description	← Double Click buttons to activate them
		7.1.1	Image name (or ID)	.tif
		7.1.2	Dimensions	approx. 3100 x 3000
		7.1.3	Resolution
		7.1.4	Bit-depth	8bit
		7.1.5	Image Location
		7.1.6	Standard image orientation
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