Name: Lactobacillus acidophilus NCFM reference proteome (pI 6-11)

Description: reference proteome of whole cell extract of Lactobacillus acidophilus NCFM

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-09-01

1.1.2 Responsible person or role

Affiliation: Technical University of Denmark

(i) Name: Susanne Jacobsen

(ii) Postal address: Department of Systems Biology
Enzyme and Protein Chemistry
Technical University of Denmark
Søltofts Plads
Building 224, room 208
2800 Kgs. Lyngby
Denmark

(iii) Email address: sja@bio.dtu.dk

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Lactobacillus acidophilus NCFM reference proteome (pI 6-11)
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 7 M urea, 2 M thiourea, 48 mM CHAPS, 200 mM bis(2-hydroxyethyl) disulfide (HED), 5% glycerol, 10% 2-propanol, and 2% pharmalyte pH 6−11

3. Gel matrix and electrophoresis protocol

3.1 Dimension details

3.1.1 Ordinal number for this dimension

First

3.1.2 Separation method employed

Isoelectric focusing (IEF)

3.2 Gel Matrix

3.2.1 Description of gel matrix

IPG strip
Denaturing

3.2.2 Gel manufacture

Gel was purchased precast.

Manufacturer: GE life sciences
Model: Immobiline DryStrip pH 6-11, 18 cm
Model number: 17-6001-97
Batch number: unknown

3.2.3 Physical dimensions

X: 180 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

linear pH 6 - 11

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 4:3

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Lactobacillus acidophilus NCFM reference proteome (pI 6-11)
  • Volume of sample: 340 µL
  • Loading buffer: 7 M urea, 2 M thiourea, 48 mM CHAPS, 200 mM bis(2-hydroxyethyl) disulfide (HED), 5% glycerol, 10% 2-propanol, and 2% pharmalyte pH 6−11
  • Volume of loading buffer: 100 µL

Loading method: cup loading.
Additional comment: IPG strips (pH 6−11, 18 cm; GE Life Sciences), were passively rehydrated (340 μL rehydration buffer) overnight (Immobiline DryStrip Reswelling Tray) at room temperature. Proteins (350 μg) from whole cell extract in 100 μL rehydration buffer were applied using cup-loading (Cup Loading Manifold; GE Life Sciences) according to the manufacturer’s instructions

3.3 Protocol

3.3.1 Buffers

(7 M urea, 2 M thiourea, 48 mM CHAPS, 200 mM bis(2-hydroxyethyl) disulfide (HED), 5% glycerol, 10% 2-propanol, and 2% pharmalyte pH 6−11 (GE Life Sciences, Uppsala, Sweden)).

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 30 V, 10 min

Hold: 150 V, 2 h

Hold: 1000 V, 30 min

Gradient: 1000-8000 V, 4 h

Hold: 8000 V, 1.75 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue supplemented with 1% DTT

4.1.3 Additional reagents

6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue) supplemented with 2.5% iodoacetamide

4.1.4 Equipment

Manufacturer: Pyrex
Model: Culture tubes
Model number: 1636/36MP

4.1.5 Protocol

Temperature: 23 °C.

Duration: 15 min.

Protocol:
Strips were equilibrated in two steps of 15 min with 5 mL equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue) supplemented with 1% DTT and 2.5% iodoacetamide, respectively.

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue supplemented with 1% DTT

4.1.3 Additional reagents

Alkylation buffer

6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue supplemented with 2.5% iodoacetamide

4.1.4 Equipment

Manufacturer: Pyrex
Model: Culture tubes
Model number: 1636/36MP

4.1.5 Protocol

Temperature: 23 °C.

Duration: 15 min.

Protocol:
Strips were equilibrated in two steps of 15 min with 5 mL equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue) supplemented with 1% DTT and 2.5% iodoacetamide, respectively.

3. Gel matrix and electrophoresis protocol

3.1 Dimension details

3.1.1 Ordinal number for this dimension

Second

3.1.2 Separation method employed

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

3.2 Gel Matrix

3.2.1 Description of gel matrix

slab gel
Denaturing

3.2.2 Gel manufacture

Gel was manufactured using the following receipe:

Reagents Quantity for 900 ml of a 12.5% gel
Acrylamide/PAGE 40% (w/v)= 281.25 ml
PlusOne Methylenebisacrylamide 2% (w/v)= 150.3 ml
Tris (1.5 M, pH 8.8)= 225 ml
10% (w/v) SDS = 9.0 ml
10% (v/v) TEMED = 1.24 ml
10% (w/v) APS = 9.0 ml
Make up to 900 ml with distilled water

3.2.3 Physical dimensions

X: 27 cm
Y: 21 cm
Z: 0.015 cm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 200 - 3 kDa

3.2.5 Acrylamide concentration

12.5 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 37.5:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: manual.
Additional comment: 1. Briefly rinse the Immobiline DryStrips by submerging them in a measuring cylinder containing SDS electrophoresis running buffer for Ettan DALT.
2. Holding one end of the Immobiline DryStrip with forceps, carefully place the Immobiline DryStrip in-between the two glass plates of the gel. Using a thin plastic spacer, push against the plastic backing of the Immobiline DryStrip (not the gel itself) and slide the strip between the two glass plates until it comes into contact with the surface of the gel.

Note: The strip should just rest on the surface of the gel.

Avoid trapping air bubbles between strip and the gel and avoid piercing the second-dimension gel with the strip.

3.3 Protocol

3.3.1 Buffers

Tris 25 mM; Glycine 192 mM; SDS 0.2% (w/v)pH 8.8

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 60 V, 1 h

Hold: 200 V, 6 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

Coomassie brilliant blue G-250

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 9 h.

Detection is described in the following reference protocol:
Citation: Electrophoresis,25, page(s) 327–1333 (2004).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

laser scanner

6.1.2 Name of equipment

Manufacturer: Microtek scanner
Model: Scan maker 9800XL
Model number: 9800XL

6.1.3 Software

Manufacturer: LaserSoft Imaging
Model: Silverfast Ai
Model number: version 6

6.1.4 Calibration

No (manual)

6.1.5 Equipment specific parameters

Default (vendor) parameters.

6.2 Acquisition Protocol

6.2.1 Image acquisition process

Default settings with 48 bit color and 16 bit grey scale scans with atleast 300 dpi

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

Lactobacillus acidophilus NCFM reference proteome (pI 6-11) (format: TIFF)

7.1.2 Dimensions

Width: 1070 px

Height: 1211 px

7.1.3 Resolution

1000 ppi

7.1.4 Bit-depth

48 bits (BrilliantColor)

7.1.5 Image location

Lactobacillus acidophilus NCFM reference proteome (pI 6-11).tif

7.1.6 Standard image orientation

Yes

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