Name: N/A
Description: N/A
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)
-
- Sample name: Filifactor alocis -PROTEOME
- Sample type: Test sample
- 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
13.2.9 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:
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: 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
13.2.9 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
camera6.1.2 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 IMAGING6.2.2 Reference to gel matrix
There is only one gel in this document.