Download - Proteomics Imaging and Analysis
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Proteomics
Scanning and Analysis
Protocols
Prepared by: Bob Morrison
STLCC-CPLS, Instrumentation Specialist
Dec 08, June 2013 (Densitometer offline)
Discover, Quality One Software
NCI Flicker Website
BioRad/UMAX GS-800
Densitometer
Gelscape Analysis Website
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Proteomics : BioRad GS-800 Densiometer Basic Features
Link to BioRad/UMAX GS-800 Users Manual (pdf)
The GS-800 calibrated densitometer delivers automatic calibration for superior accuracy, resolution, and
image quality. With a 30 x 40 cm (11 x 16") imaging platen, 12-bit data collection, and resolution down to
36.3 µm
Automated self-calibration for accurate quantitation
Variable scanning resolution for optimum image acquisition
12-bit (4,096 gray levels) data collection for greater linearity and sensitivity
Dynamic range from 0 to 3.0 OD
Scanning white light (400–750) nm illumination for high sensitivity
Transmittance and reflectance collection for imaging a wide variety of samples
Powerful, user-friendly Quantity One software for image acquisition and data analysis
Sealed platen for imaging of wet samples
Modified transparent lid for safely imaging samples of variable thickness
Provides quantitatively accurate, high-quality imaging to detect and resolve even the faintest
bands. With user-selectable scan resolution down to 36.3 µm, you can preview and scan your
samples in seconds. Variable resolution also allows you to control both the scanning time and
image file size.
High sensitivity. The color CCD camera can scan in red, green, or blue, and scans virtually any
sample (1-D and 2-D gels, colorimetric dot and slot blots, film-based chemiluminescent blots,
autoradiograms, slides, and photographs) at the ideal wavelength for greatest sensitivity and
accuracy.
Has both transmittance and true reflectance capabilities that allow accurate scans of both
transparent and opaque samples. The advantage of true reflectance scanning is the accurate
detection and analysis of molecules on the surface of membranes, rather than molecules
distributed throughout the membrane matrix. True reflectance provides the most accurate
detection and analysis of dot blots, slot blots, and other electrophoretic blots without
quantitation errors.
Densitometer Offline June 2013
The Densitometer software does not run on the
standard Windows operating system used on newer
PCs, therefore it has been disconnected from the
Imager host PC.
As a backup, an older PC is stored below in the
cabinet and can be configured for use of the
Densitometer. Please contact Bob Morrison in R123
or by phone (314-971-3795) for assistance in setting
this up.
Proteomics: Densitometer-Acquire Image- Quality One
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1. Turn on Densitometer/Scanner and
wait for both lights to show steady
green- this is the ready state.
2. Turn on Host computer, logon as
Operator/SMET
3. Select Quality One to scan (acquire)
and image
Link to BioRad Quantity One Software Users Manual (pdf)
Proteomics: Quality One-Select Scanner
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1. Choose Select Scanner on the
Volume Quick Guide menu
2. Select GS-800 as scanner type
Proteomics: Quality One- Scan-Step I-Select Image Type
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1. Step I, Select image and then stain types
2. Step II, Preview Scan and wait for image
outline to appear.
3. Hit STOP if image preview fails to appear
Proteomics: Scan- Step II-Select Scan Area, Step III –Select Resolution
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1. Drag box to outline scan image
2. Step III, change scan resolution in
pop-up box if required
Proteomics: Scan- Acquire Image, wait for scan to complete
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Scanning; Wait for Scan in to proceed
Proteomics: Quality One Densitometer- Scan-Save Image
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To save scanned image select File, then
Save-as, then change filename, and
Save
Proteomics: Quality One - Image-Analysis-Lanes-Volumes
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Follow this link to clarify lanes and identify purity on your scanned image.
Link to Bio-Rad “Quality One” Lane Analysis Tutorial (pdf)
Follow this link to identify volumes (quantitation) for unknowns and
standards on your scanned image and to generate reports of both:
Link to Bio-Rad “Quality One” Volume Analysis Tutorial (pdf)
Lanes
Volumes
Proteomics: Image-Gel Issues (Examples)
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Follow this link to see examples and causes of common gel issues.
Link to Bio-Rad “Gel Clinic” pdf)
Proteomics: SDS_PAGE
Examples
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Proteomics: Gelscape- Enter Username, Password
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http://www.gelscape.ualberta.ca:8080/htm/index.html
Username: Ktoal
Password: 17gelscape
Link to Gelscape
U. Of Alberta Canada
Proteomics: Gelscape-Window Size-Annotate & View Panel
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Adjust Window size to
Enlarge annotation panel
Proteomics: Gelscape-Browse to find your Image
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Browse to locate
your image (gif, jpg)
Proteomics: Gelscape- Morph & Compare
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Browse to locate
your image (gif, jpg)
Proteomics: Gelscape –GelBank- Other image sources
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Search in Gelscape or
other databases
Proteomics: Gelspace- Access other 2D images
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Link to NCI
Flicker Website
Proteomics : BioRad Densiometer; 2D Gel Flicker Website
Link to
http://www.ccrnp.ncifcrf.gov/flicker/indexFrmToc.html
Proteomics: 1D PDQuest-Help-Register-Enter Password 1/26/09 RGM
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Proteomics: Gel Electrophoresis Background
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Gel Electrophoresis: Gel Electrophoresis is a separation technique that is used to separate macromolecules such as nucleic acids
or proteins on the basis of size, electric charge, and other physical properties.. It can be performed within one dimension, two
dimensions, or in a capillary. The sample is mixed into a buffer, and run on gels. Electrophoresis separations are usually carried out
on gels made of agarose or polyacrylamide. These gels are chemically inert, so they will interfere little with the molecules. When
electrophoresis is complete, the gel is stained to make the proteins visible. Common dyes used are Coommasie blue or silver
staining. This method of protein separation and identification is useful because very little protein is needed to determine a difference
in a protein. Distinct spots can be found with as little as 0.1 mg of protein when stained with Coommasie blue and even less (0.02
mg), is need when silver staining. Other methods of staining are Fluorescent dyes, and zinc or copper staining. Some drawbacks to
this technique are that it is time-consuming and the purity of your protein sample will affect your results.
One-Dimensional: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) - this method separates based on the
mass of the molecule. Sodium dodecyl sulfate (SDS) is a detergent that breaks up the interactions between proteins. The proteins
are dissolved in SDS and then electrophorised. The smallest molecules move through the fastest, while larger molecules take longer
and result in bands closer to the top of the gel.
Isoelectric Focusing(IEF) - this method separates based on the isoelectric point(pI) of the protein. The isoelectric point is when the
protein carries no net charge. The protein sample is applied to an immobilized pH gradient(IPG) strip. IPG strips come available over
different lengths and pH ranges. As the sample is electrophorised, the proteins will migrate toward either the anode or cathode,
depending on charge. The proteins will stop when they reach their respective pI, which results in a band. The band corresponds to a
protein.
Two-Dimensional : 2D Electrophoresis is a technique that blends the two methods listed in One-Dimensional analyses. It is a very
efficient separation technique for proteins. The protein sample is first run on an IPG strip, which after reaching completion is placed in
either a horizontal or vertical SDS gel. This technique results in gels that contain spots. Each spot on the gel corresponds to a
different protein. Then the gels are stained similarly as to 1D analysis. 2D Electrophoresis is widely used, and certain methods of it
can be coupled with mass spectrometry in order to identify proteins.fgfrgcfsdfdfs
Capillary: Capillary Electrophoresis is very similar to 1D or 2D Electrophoresis, except it is performed within the small space of a
capillary tube. This is advantageous in many ways. The heating that takes place due to high voltage loads on slab gels can have a
negative effect on the separation of the proteins and the use of capillaries lessens said heat build-up. Also, because the gel will not
need to be handled, it can allow one to use liquid polymers for separation, and can be replaced between runs. Automation is also
much more possible with this technique, lessening the time, and making reproducible results.
Retrieved from "http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/What_is_Electrophoresis%3F"
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Proteomics : Themo Chiller for Gel Supercooling Coil
(example only not purchased)
NESLAB RTE-7 Digital Plus Refrigerated Bath
271103200000-00i
$2,957.00
The RTE-7 is the perfect partner for most CCC applications requiring temperature regulation.
This refrigerated/heated circulating bath offers the highest cooling capacity per dollar, a small
footprint to conserve valuable bench space, and a 2 year warranty for peace of mind.
Temperature Range :-25°C to +150°C
Cooling Capacity :500 Watts at 20°C fluid temperature
Pump Flow :15 liters per minute maximum
Pump Pressure:16 feet maximum
Pump Type:Force and Suction
Heater Wattage :800 Watts (2000 Watts 230 volt models)
Temperature Stability :+/-0.01°C
Reservoir Volume:7 liters Reservoir
Dimensions (WxLxD) :6.6" x 7.2" x 6" (16.8cm x 18.3cm x 15.2cm)
Unit Dimensions (HxWxD) :23 5/8" x 9 1/4" x 17 1/2" (60cm x 23.5cm x 44.5cm)
Shipping Weight :63 lbs. (28.6 kgs)
The Thermo Scientific NESLAB RTE-7 7 liter refrigerated circulating bath offers great value
for either external circulation or in-bath applications:
· Large work openings for big or multiple samples
· Advanced features to further optimize your system available with the Digital Plus
· Wide temperature range and high cooling capacities (500W) to meet your application needs
(down to -25°C)
The super cooling coil and a water re-circulator maintain constant buffer
temperature and prevent buffer depletion during native enzyme, high-
intensity, or overnight transfers