2 D Electrophoresis

Starting/Decisions to be made


Contact: Hyuk-Kyu Seoh
Rm (lab): PSC 537

Rm (office): PSC 521
Tel: (404) 413-5379

Click on 2D analysis tab again to see all available 2D electrophoresis guides.

2D electrophoresis manuals:
Sample Prep
2D Cleanup
2D Quant


Starting/Decisions to be made topics:
Equipment for 2-D Gels
Time Sequence for 2-D Gels
Detection and Analysis Decisions

GE Amersham Manuals:
Amersham 2-D electrophoresis Manual
Ettan DIGE System User Manual

Table of Detection choices - cost, Pros and Cons
Amount of Sample Needed
Protein Cleanup
Protein Concentration

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.

Most of the basic information came from the 2-D Electrophoresis principles and methods manual from Amersham. This book is VERY good – not only does it give you protocols, but also tells you about principles that are used in 2-D electrophoresis, the reasons for using specific chemicals and choices for sample prep and cleanup. I have included the link to this manual. There is also a Trouble shooting section at the end of each chapter.

Equipment for 2-D Gels:
Equipment for 1st-dimension:
IPGphor Isoelectric Focusing System
IPGphor Rehydration Tray
IPGphor Cup Loading strip holder (can choose 7, 11, 13, 18 or 24 cm)
IPG strip (Immobiline DryStrip gels) - can choose 7, 11, 13, 18 or 24 cm; many choices of pH ranges – see Supplies.
Equipment for 2nd dimension:
Ettan gel caster
Ettan DALT six system: separation unit (can hold up to 6 gels); power supply; gel casting cassettes.
SE600 Ruby gel electrophoresis unit

Time Sequence for 2-D electrophoresis:
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.
• Strip hydration and IEF can be done separately. Do hydration o/n and IEF the next day
• 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 –scan using Amersham Typhoon (Cy Dye’s only; 40 min./gel, Should be done on the same day.
• Deep Purple/SYPRO Ruby, Krypton Stain, Coomassie - Add another day; these either stain or fix o/n
before scanning
Day 3
• Finish staining if needed.
• Scan if needed
• Analysis – DeCyder/ImageMaster program
• Spot picker
• Trypsin digest
o/n
Day 4
• Finish Trypsin digest
• Zip tip if needed
• Spot on MALDI plate
• MALDI run

Day 5
• MALDI run Analysis

Notes:
• Always wear nitrile gloves when handling everything from sample prep until loaded on MALDI plate for analysis.
• Use high quality reagents and ddH2O
• See each section for additional precautions and see below for how to clean the various components.


Detection and Analysis Decisions.
Before starting, you have to decide how you want to detect your proteins of interest. See table below for costs/gel, Pros and Cons of each detection method. You must also decide how your results need to be analyzed and whether the results must be CFR part 11 Complient (you will need to use CyDyes and the Decyder software). 
1. Analytical vs. Preparative gels.  If you use CyDyes or fluorescent stains, you may not have enough protein in the spot to get a good protein ID – you load much less protein on the gel.  If this is the case, you will need to do analysis using whatever  detection method of choice and then run another gel with much more protein and stain with Coomassie.  Shelby has used Deep Purple for her Preparative gels.  She felt that she was missing some important spots that just were not picked up by Coomassie.  Each lab/project will need to make this decision based on what you are looking for.
2. CyDyes. Before doing IEF, the control proteins are labeled with either Cy3 or Cy5 and the sample proteins are labeled with Cy5 or Cy3(dye swapping, see  #3). You can also pool the 2 and label with Cy2. All 3 of these are combined and run on a single 2D gel (1st and 2nd Dimension). The gel is then scanned at 3 different wavelengths and the analysis software combines the 3 images and looks for differences using DeCyder analysis software.  Reasons for including a pooled sample labeled with Cy 2, see Sample Prep section.  Highly recommended.
3. Dye Swapping.  Don’t label all controls with 1CyDye and all samples with the other.  The MW of each dye is slightly different and swapping Dyes will decrease this variable.  As a result, the statistics in Decyder is much better when Dye swapping is used. 
4. Fluorescent Stains. Staining is done after 2D gel has run. Keep in mind that Deep Purple is more expensive than Krypton, but since it is more sensitive you would not need as much protein. This could be especially important with rare protein samples.
5. Generic Stains. Coomassie Blue and Silver Staining are done after the 2D gel has run.
6. CFR part 11 Complient. CFR part II was established by the FDA to provide guidelines for how data is stored and processed. ImageMaster analysis software is NOT CFR part 11 complient and Decyder analysis software IS CFR part 11 complient. If you need to be CFR part 11 complient, you have to use CyDyes and Decyder software. All other dyes must be analyzed on ImageMaster.


 Table of Detection Choices

Detection choices
 Cost/gel
Pros
Cons
CyDyes
   
400 pmole/sample; 2 dyes
$120.00
Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots
400 pmole/sample; 3 dyes
$180.00 
Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots
200 pmole/sample 2 dyes
$60.00 
Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots
200 pmole/sample 3 dyes
$90.00 
Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots
SYPRO Ruby
$72.00 
Sensitive, but not as sensitive as CyDyes   Control and sample have to run on separate gels; takes an additional day to process; can’t visualize protein spots, so have to use Typhoon
Deep Purple  
$70.00 
Sensitive, but not as sensitive as CyDyes  Control and sample have to run on separate gels; takes an additional day to process; can’t visualize protein spots, so have to use Typhoon
Pierce Krypton
$36.00 
Sensitive, but not as sensitive as CyDyes or Deep Purple/SYPRO Ruby  Not as sensitive as Deep Purple, but background was MUCH better.  Control and sample have to run on separate gels; takes an additional day to process; can’t visualize protein spots, so have to use Typhoon
Coomassie Blue
$6.43
Cheap; visualize spots so can cut them out by hand or use SpotPicker   Not nearly as sensitive as above; Control and sample have to run on separate gels; takes an additional day to process 
Invitrogen Simply Blue
$10.00
Cheap; visualize spots so can cut them out by hand or use SpotPicker   Not nearly as sensitive as above; Control and sample have to run on separate gels; takes an additional day to process 
Silver Staining
$13.50
More sensitive than Coomassie; visualize spots so can cut them out by hand or use SpotPicker   Not as sensitive as CyDyes and Fluorescent dyes; Control and sample have to run on separate gels; takes an additional day to process 
 
 
 
 

Amount of Sample Needed.
Amount depends on what detection method will be used. Listed below are the amounts that Rollin and Anupama use. These amounts should be used as a guide only; each lab/system will need to have amounts optimized for your specific experiment.
1. 50 ug for Cy Dyes
2. 30 – 40 ug for SYPRO Ruby or Deep Purple
3. 200 – 400 ug for Coomassie
4. 100 ug for Western blotting
5. 200 ug for Silver Stain
The manual also has a table (p. 55) with protein sample load recommendations. The amount varies depending on strip length, pH of strip and whether the gel is analytical or preparative. It only has amounts of protein for Silver stained and Coomassie gels though.

*If you do cup loading, do not exceed 150 ug/150 ul sample solution.  If you go over this concentration, you could get protein precipitation at the sample cup.

2 Protein Cleanup recommendations from Roberto:
1. 2D clean up Kit from GE
2. Millipore filter UFV to concentrate and clean up proteins

Determine Protein concentration.
1. GE 2D Quant kit is able to get an accurate protein concentration even in the presence of DTT, thiourea and EDTA.
2. Crude OD using spectrophotometer
3. BCA Protein Assay Kit from Pierce. Cheaper than using the GE 2D Quant Kit

 

 


 


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


 

 


 

 

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