Proteomics  



Contact: Debby Walthall
 or Hyuk-Kyu Seoh
Rm: NSC 338 or 516
Tel: (404) 413-5363 or (404) 413-5379



Proteomics Service Request Form
Charges : 
 

Gel Services:
Small gel format 

$80.00/gel (includes 2D gel set-up, supplies, scan, analysis and spot picking)
 
does not include cost of staining ($10.00 - $180.00 per gel)
Gel Services:
Large gel format 
$120.00/gel (includes 2D gel set-up, supplies, scan, analysis and spot picking)
 
does not include cost of staining ($10.00 - $180.00 per gel)
MALDI MS: 
Less than 5 samples
 
$20 per spot - includes gel pick and in-gel digest ($3.00 per spot); ZipTip cleanup ($3.00 per spot); spotting, MS and MS/MS analysis ($14.00 per spot)
MALDI MS: 
More than 5 samples
 
$60.00 setup and $8.00 per spot - includes gel pick and in-gel digest ($3.00 per spot); ZipTip cleanup ($3.00 per spot); spotting, MS and MS/MS analysis ($2.00 per spot)
This technique sorts proteins according to two independent properties in 2 steps: the first-dimension step, isoelectric focusing (IEF)  ,separates proteins according to their isoelectric points (pI); the second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to the molecular weights (MW). Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Basic information for 2-D Electrophoresis principles and methods manual from can be found in the Amersham 2-D electrophoresis Manual. There is also a Trouble shooting section at the end of each chapter. There are numerous other manuals on the same page that should help you get started.

Time sequence for an Experiment
Supply List

Related Website:
General information about 2D electrophoresis


2D electrophoresis Manuals:
Starting/Decisions to be made
Sample Prep
1st Dimension  
2nd Dimension  
Visualization 2D gels
Scanners - Typhoon and PDSI (Typhoon.pdf) 
ImageMaster Analysis software
Decyder Analysis Software
Ettan Spot picker (Spot picker.pdf) 
Trypsin in-gel digest
Sample desalt/cleanup - ZipTip protocol

Sequence for 2-D electrophoresis:
Following is a sequence if you do a run as quickly as you can. After you have scanned your gel, you could save the gel until the next day to pick spots. You could also wait to process the spots for a few days, just remove the H2O from the well. Don’t wait much longer or the protein will start to break down.
Sample prep – varies depending on protein source and sample needs to be soluble.
Day 1
• IPG strip hydration – provided dry and must be rehydrated with the appropriate additives prior to IEF.
• IEF – performed on a flat bed system (IPGphor IEF system) at very high voltages with active temp. control. Time of run depends on purity of protein sample, usually 24 hrs.
• Strip hydration and IEF can be done either separately or at the same time. Do hydration o/n and IEF the next day. Strip can also be frozen o/n.
• Pour 2nd dimension gel.
Day 2
• CyDye labeling if needed.
• IPG strip equilibration – in SDS-containing buffer prepares the sample for the 2nd dimension separation.
• SDS-PAGE – the strip is placed in the 2nd dimension gel. Time of run is usually 4 –5 hrs, it depends on how many gels you run. This can also be done at low power o/n.
• Visualization – Either staining/destain (o/n) or scan using Amersham Typhoon (Cy Dye’s only; 40 min./gel, Deep Purple/SYPRO Ruby) or Personal Densitometer SI (Coomassie/Deep Purple/SYPRO Ruby). Should be done on the same day.
• Add another day for SYPRO Ruby – stains o/n
Day 3
• Analysis – DeCyder/ImageMaster program
• Spot picker
• Trypsin digest o/n
Day 4
• Finish Trypsin digest
• Zip tip if needed
• Spot on MALDI plate
• Data collection and analysis

This information is given as a guide to the facilities and instrumentation available in the DNA/Protein Core facility at Georgia State University. If you have any concerns or thoughts about the content of this website please contact: John Houghton (404) 413-5390