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COBRE Core Facilities

COBRE CORE Facility

Located in J. Bennett Johnston Builidng, room 435

The COBRE Core facility is dedicated to providing state-of-the-art equipment and expertise to the investigators in the Cancer Genetics Program. This involves the development of protocols for critical assays needed by our investigators, as well as helping them interface with existing Cores.

For information on next generation sequenceing at Tulane Cancer Center please go to:

http://tulane.edu/som/cancer/nextgenseqanalysiscore.cfm

2D Electrophoresis and 2D DIGE for Analysis of Protein Samples

The COBRE Core Proteomics Lab is capable of separating protein samples via traditional 2D or 2D DIGE as well as transferring gels to membranes for Western analysis. Assistance is also provided with sample preparation. The lab is equipped to run mini- to large-format gels and has software for spot detection and analysis, and generating lists for spot picking using the Ettan Spot Handling Workstation (LSU Proteomics Core).

Equipment

  • IPGphor II isoelectric focusing system
  • ETTan DALTsix Electrophoresis Unit
  • Typhoon Trio + Variable Mode Imager
  • PDQuest 2D Software
  • DeCyder 2D Software

Traditional 2D

Protein samples (one sample/gel) are separated in the first and second dimension and then stained with a variety of total protein stains using visible or fluorescent dyes (Coomassie, SYPRO Ruby, Ponceau S, etc) or with protein-specific dyes (e.g. phosphoproteins or glycoproteins) Samples run on strips up to 18cm long (in first dimension) can be transferred to membranes for Western analysis (2D Gel Western Blotting)

2D DIGE (DIfference Gel Electrophoresis)

  • Protein samples are labeled with fluorescent dyes prior to electrophoretic separation
  • CyDye DIGE fluors allow for multiplexing—separation of up to three different samples within the same gel. CyDye DIGE Fluors are:
    • Size and charge matched. The same protein from different sampleswill migrate to the same position regardless of the dye used
    • pH insensitive
    • Spectrally resolvable
    • Highly sensitive and bright
    • Photostable
  • DeCyder 2D Software designed specifically for DIGE applications. DeCyder utilizes a proprietary co-detection algorithm that permits automatic detection, background subtraction, quantitation, normalization, and inter-gel matching of the fluorescent images

Comet Assay for DNA Damage and Repair

The single cell gel electrophoresis or Comet Assay is widely used to investigate DNA damage and repair in different cell types in response to DNA damaging agents. The Comet Assay has also been used in applications such as human biomonitoring, genotoxicology, and ecological monitoring. The COBRE Core lab can process cell samples derived from adherent or suspension cultures as well as tissue and is equipped with Comet Scoring software for quantitative analysis.

Assay Principle

  • Denatured, cleaved DNA fragments will migrate out of the cell under the influence of an electric field
  • Cells appear as a “comet” with a distinct head, comprised of intact DNA and a tail consisting of damaged or broken pieces of DNA
  • Assessment of DNA damage achieved by evaluation of the DNA comet tail shape and migration pattern

Advantages of the Comet Assay

  • Simple/Inexpensive
    • Easy sample preparation and small amount cells needed
    • Equipment required includes horizontal box and microscope (fluorescence or standard light)
  • Rapid
    • Assay time < 4 hrs
  • Sensitive
    • Various classes of DNA damage (SSBs and DSBs) can be detected
    • Lesion-specific enzymes can be used to detect specific types of DNA damage
    • Assay can be modified for measurement of DNA cross-linking and alkylation

Analysis

  • CometAssayIV (Perceptive Instruments) comet scoring software
  • Click & Score automatic scoring method marks cells to prevent them from being scored twice
  • Results window can be hidden for blind scoring
  • Measures Olive Tail Moment, Head and Tail Lengths, Head and Tail % intensity, Tail Migration, and more

COBRE CORE Charges - Proteomics

CHARGING UNIT

AMOUNT

per sample

 

$4.50

2-color DIGE

per gel

$85.00

3-color DIGE

per gel

$127.50

7/11/13cm IEG/2D

per gel

$18.50

18 or 24cm IEF/2D

per gel

$37.00

Coomassie Staining

per gel

$24.00

SYPRO Ruby staining

per gel

$85.00

Gel Scanning and Analysis

per hour

$55.00

 

COBRE CORE Charges - Comet Assay

CHARGING UNIT

AMOUNT

sample processing (Alkaline, Neutral)

per sample

$5.00/sample for first 5 samples, $3.75/sample for each additional sample

imaging and analysis

per sample

$15/sample for first 5 samples (75 comets scored/per sample), $10/sample for each additional sample


For sample submission please use the following:
Proteomics Sample Submission Form
Comet Assay Sample Submission Form

For questions/service contact:
Melody Baddoo
1324 Tulane Avenue, JBJ/425
New Orleans, LA 70112
504-988-0568
mbaddoo@tulane.edu

 

 

 

 

 

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1430 Tulane Ave, New Orleans, LA 70112 504-988-5263 medsch@tulane.edu