Gel: Filifactor alocis-Total proteome (html display)

Name: Filifactor alocis-Total proteome

Description: Flifactor alocis - 2D-PAGE and MS/MS analysis of the total cell lysate

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2011-10-10

1.1.2 Responsible person or role

Affiliation: FLETCHER LAB , SCHOOL OF MEDICINE ,LOMA LINDA UNIVERSITY

(i) Name: WILSON ARUNI, FRANCIS ROY and HANSEL .M.FLETCHER

(ii) Postal address: DIVISION OF MICROBIOLOGY AND MOLECULAR GENETICS
SCHOOL OF MEDICINE
LOMA LINDA UNIVERSITY
LOMA LINDA -CA-92354,USA

(iii) Email address: waruni@llu.edu

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

1. Sample name: Filifactor alocis -PROTEOME
2. Sample type: Test sample
3. URL: http://iai.asm.org/cgi/reprint/79/10/3872

2.1.2 Loading buffer

standard loading buffer for 2D gel electrophoresis

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 manufactured using the following reference protocol:

Citation: Poznanovic, S., G. Schwall, H. Zengerling, and M. A. Cahill. 2005. Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 26:3185–3190.,26, page(s) 3185-3190 (2005).
URL: http://iai.asm.org/cgi/reprint/79/10/3872

3.2.3 Physical dimensions

X: 7 cm
Y: 3 cm
Z: 1 cm

3.2.4 Physiochemical property range and distribution

linear pH 3 - 10

3.2.5 Acrylamide concentration

10 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 3.2:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

3.2.9 Sample application

Lane 1

Sample: Filifactor alocis -PROTEOME
Volume of sample: 5 µL
Loading buffer: standard loading buffer for 2D gel electrophoresis
Volume of loading buffer: 15 µL

Loading method: well loading.

3.3 Protocol

3.3.1 Buffers

2D-REHYDRATING BUFFER

3.3.2 Electrophoresis conditions

Running temperature: room temperature (no cooling device).

Hold: 300 V, 3 h

Hold: 3500 V, 5 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

EQUILIBIRATION BUFFER

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: BIORAD
Model: BIORAD-PROTEAN ISOELECTRIC FOCUSING
Model number: PROTEAN

4.1.5 Protocol

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 reference protocol:

3.2.3 Physical dimensions

X: 7 cm
Y: 5 cm
Z: 1 cm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 10 - 250 kDa

3.2.5 Acrylamide concentration

10% %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 3.2:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

3.2.9 Sample application

Loading method: well loading.

3.3 Protocol

3.3.1 Buffers

LOADING BUFFER-1

3.3.2 Electrophoresis conditions

Running temperature: room temperature (no cooling device).

Hold: 140 kV, 50 min

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

COOMASSIE SIMPLY BLUE STRAINING

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

Manufacturer: BIORAD
Model: X CELL SURE-LOCK
Model number: BIORAD

5.1.5 Direct detection protocol

Detection is described in the following reference protocol:
Citation: Poznanovic, S., G. Schwall, H. Zengerling, and M. A. Cahill. 2005. Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 26:3185–3190.,26, page(s) 3185-3190 (2005).
URL: http://iai.asm.org/cgi/reprint/79/10/3872

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

camera

6.1.2 Name of equipment

Manufacturer: UVP
Model: LAUNCH VISION WORK LS
Model number: UVP

6.1.3 Software

Manufacturer: UVP
Model: LAUNCH VISION WORKS LS
Model number: LAUNCH VISION WORKS LS

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Default (vendor) parameters.

6.2 Acquisition Protocol

6.2.1 Image acquisition process

DEFAULT COMPANY SETTINGS FOR 2D PAGE IMAGING

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

FA-2D (format: .psd)

7.1.2 Dimensions

Width: 1000 px

Height: 1000 px

7.1.3 Resolution

1000 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

FA-2D(mini).psd

7.1.6 Standard image orientation

Yes