2 D Electrophoresis

Imaging/Visualization


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

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


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


Imaging topics:
Supply List
Table of Detection choices - Pros and Cons
Table with Detection choices - company, catalog #'s and cost
SYPRO Ruby Staining
Deep Purple Staining


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


Pierce Krypton Staining
Coomasie Blue Staining
Invitrogen Simply Blue
GelCode Blue
Silver Staining

There are multiple documents each claiming that SYPRO Ruby or Deep Purple is more sensitive. You decide – cost is almost the same but Deep Purple takes 1 day less to process

2-D gel Visualization
Now that your gels are ready, there are various choices for visualizing the peptides spots. There are a number of staining methods to stain the peptides (Coomassie staining, Silver staining, SYPRO ruby staining, Deep purple staining, new Krypton staining, among others). You can use the Typhoon 9400 (NSC 438), which can detect fluorescence with CyDyes, Deep Purple, SYPRO Ruby and Krypton stains. You can also use the Personal Densitometer SI (PDSI) for Coomasie Blue. Immediately scan CyDye gels. If not using CyDyes, proceed to staining method of choice.

Notes:
• Staining works best in plastic trays with lids, not glass.
VERY IMPORTANT. Speckling on gels using Deep Purple or Krypton Protein Stain can be a problem. Either use different trays for each of these OR rinse tray very well with H2O, EtOH and Methanol. Especially do not use Coomassie stained trays for Deep Purple or Krypton Protein Stain.
• Staining works best in plastic trays with lids, not glass. • Keep trays separate for each type of staining. Speckling can occur when trays used for Coomassie are used for Deep Purple or SYPRO Ruby
• Multiple documents each claiming that SYPRO Ruby or Deep Purple is more sensitive (see Amersham application note #18-1177-44; Compariston of Deep Purple Total Protein Stain and SYPRO Ruby in 1-D and 2-D gel electrophoresis). You decide – cost is almost the same but Deep Purple takes 1 day less to process.
• See table for costs, Pros and Cons of the different methods.
• Krypton Protein Stain is a new stain from Pierce and can also be purchased from Fisher.
• The Coomassie method is from Introgen’s iprotocol.
• Some labs use the PROTSIL1 ProteoSilver Stain kit from Sigma – cat.#: PROTSIL1-1KT.

Read the Amersham manual for information about Visualization, not much detail though.
• Various available techniques available to see the peptides, including pros and cons of each technique.
• Analysis of results
• Standardization of results
• Further analysis of results

Typhoon/PDSI -> SpotPicker
Separate guide for Typhoon and for PDSI.
You can use any fluorescent dye (CyDyes, SYPRO Ruby, Deep Purple or Krypton) and scan the gel image into the Typhoon or PDSI. For CyDyes, you can use the DeCyder software to generate a picklist (text file). For other fluorescent dyes, you can use ImageMaster to generate a picklist. This picklist is sent to the SpotPicker so it knows the X,Y and Z of the spots you want. Don’t forget you must have a reference marker on the gel that the Spotpicker uses to align the picker head. You can either place the reference marker on the glass plate before pouring the gel (works best) or you can attach the marker to the back of the gel sandwich after the run. The marker absolutely cannot move once the gel is scanned. Otherwise when the Spot Picker uses the moved marker to align itself with the gel, the location of the spots will be changed. If the marker moves, you will need to rescan and generate a new picklist.

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, so have to do post stain and use Spotpicker
400 pmole/sample; 3 dyes $180.00  Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots, so have to do post stain and use Spotpicker
200 pmole/sample 2 dyes $60.00  Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots, so have to do post stain and use Spotpicker
200 pmole/sample 3 dyes $90.00  Very sensitive; can run control and sample on the same gel Expensive; can’t visualize protein spots, so have to do post stain and use Spotpicker
SYPRO Ruby $67.00  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 Spotpicker
Deep Purple   $67.20  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 Spotpicker
Pierce Krypton $35.00  Sensitive, but not as sensitive as CyDyes   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 Spotpicker
Coomassie Blue $1.00 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 
Invitrogen Simply Blue $1.72 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 $16.94 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 
     


Table of Detection choices - company, catalog # and cost
Stain
Company
 Catalog #
Cost/order
Amt./order
Amt./gel
CyDye 2
GE/Amersham
25-8008-60
$1526.00
10 nM
400 pM or 200 pM
CyDye 3
GE/Amersham
25-8008-61
$1526.00
10 nM
400 pM or 200 pM
CyDye 5
GE/Amersham
25-8008-62
$1526.00
10 nM
400 pM or 200 pM
SYPRO Ruby
Invitrogen
S12001
$67.00
200 ml
200 ml
Deep Purple
GE/Amersham
RPN6305
$168.00
5 ml
200 ul
Krypton
Fisher (Pierce)
PI46628(46628)
$82.70
20 ml
10 ml
Coomassie Blue
BioRad
161-0400
$18.00
10 gm
100 mg
Simply Blue
Invitrogen
LC6065
$100.00
3.5 L
60 ml
GelCode Blue
Pierce 
24590
$55.00
500 ml
50 ml
ProteoSilver
Sigma
PROTSIL1-1KT
$135.50
1 kit
1/8th kit
   
     

CyDyes
See 1st Dimension.
Sample Prep for CyDyes is done before 2-D run.

SYPRO Ruby Staining
• 100 ml for 1 mid-size gel
• Rollin uses 400 ml for his full size Ettan DALT gels.
• You need enough stain to cover gel.
• Gels can be left in fix o/n.
• Gels can be stored in SYPRO Ruby stain indefinitely.
• Keep gel covered and out of light during staining and wash steps.

Reagent and Solutions
Fixing solution
7% acetic acid / 50% methanol – 1.2L/gel – 1L

70ml acetic acid
500ml methanol
430ml ddH2O

Stain solution
SYPRO Ruby stain

Wash Solution
10% methanol, 7% acetic acid - 1L

100 ml methanol
70 ml acetic acid
830 ml ddH2O

Protocol (for full Manual)
1. Thoroughly clean and rinse (ddH2O) staining containers. Then rinse with EtOH
2. Fix with gentle (25rpm) shaking in fixing solution. 500 ml for 30minutes – basic protocol; 500 ml for 15 min. rapid protocol.
3. Remove gel, pour out fixing solution, rinse container then fill with 100ml SYPRO Ruby and re-insert gel. Basic protocol: incubate with gentle shaking (40rpm) overnight protected from light. Rapid protocol: microwave 30 seconds; agitate 30 seconds; microwave 30 seconds; agitate 5 minutes; microwave 30 seconds; agitate 23 minutes.
4. Transfer gel to a clean container. Wash gels with 100 ml of Wash Solution with gentle agitation for 30 minutes. Rapid method same.
5. For both methods, rinse gel with ddH2O a minimum of 2 times for 5 minutes each with agitation.
6. Store gel between 2 pieces of glass in 0.75% ethanol until scanning

Deep Purple Staining
• Deep Purple Total Protein Stain. Instructions. RPN6305PL Rev E 2004.
New protocol May 2006 using boric acid instead of carbonate/bicarbonate buffer and citric acid to fix instead of acetic acid.
• Use this protocol instead of the one that comes with Deep Purple order.
• Besides taking less time, this protocol is much more sensitive and does not need to be done in the dark.
• New protocol is below.
• 15% EtOH in Fixing solution, washing solution and acidification solution can be replaced with 30% methanol. Whichever you decide on EtOH or methanol, use the same for all 3 solutions.
• There is a protocol in the May 2006 manual for Non-fixing of 1D gels.

Reagents and Solutions
Fixing solution
15% EtOH, 1% citric acid – 1L

150 ml EtOH
10 gm citric acid
850 ml ddH2O

Staining solution
100 mM Sodium borate, pH 10.5 – 10.8 – 1L

6.2 gm boric acid
800 ml ddH2O
pH to 10.5 with NaOH, then bring up to 1L
Combine 2 ml of Deep Purple stain + 500 ml staining solution
remove stain from -20C and incubate at room temp for 2 min
shake concentrate briefly

Wash solution
15% ethanol – 1L

150 ml EtOH
850 ml ddH2O

Acidification
15% EtOH, 1% citric acid – 1L

150 ml EtOH
10 gm citric acid
840ml ddH2O


Standard Protocol
1. Amounts listed below are for the large Ettan DALT gels. Adjust down if your plates are smaller.
2. Thoroughly clean and rinse (ddH2O) staining containers. Do not use any containers that have been used for Coomassie staining.
3. Prepare fixing solution.
4. Place gels in 1L of fixing solution and incubate with gentle rocking for at least 1 hour. This time can be extended to o/n. O/N fixing can reduce the background.
5. Remove gel, rinse container and incubate in Staining Solution. Stain for 1 hour with gentle agitation (40rpm) Time can be extended to 4 hours without adverse affects.
6. Remove gel, rinse container, and place gel in 1L wash solution with gentle agitation for at least 30 min.
7. Remove gel, rinse container and place gel in 1L acidification solution and agitate gently for 30 min.
8. Storage. Gel can be stored in the acidification solution containing 1:200 diln. of Deep Purple. Or recycled staining solution can be used. If you used recycled staining solution, adjust the pH to 2.4 by adding ~5 gm citric acid/1L solution; filter the solution before using for long term storage.
9. Scan gel using the green laser (532nm) and emission filter 560LP (PMT voltage will have to be adjusted according to the protein load if saturation is an issue…start with 550…do not go above 600).

Pierce Krypton Protein Stain
• Almost as sensitive as above stains, except cheaper and lower background.
• Compatible with MS
• Rapid method can be done in 30 min.
• Use enough solution to cover gel.
• Problems with speckling. Do not use staining trays that have been used for Coomassie staining; wash trays very well.

Reagents and Solutions
Fixing solution
40% EtOH, 10% acetic acid – 1L

400 ml EtOH
100 ml acetic acid
500 ml ddH2O

Staining solution
10 X stock

10 ml 10X stock
90 ml ddH2O

Destaining solution
5% acetic acid

50 ml acetic acid
950ml ddH2O

Standard Protocol
1. Thoroughly clean and rinse (ddH2O) staining containers
2. Prepare fixing solution
3. Place gels in 1L of fixing solution and incubate with gentle rocking for at least 30 minutes.
4. Pour off fixing solution and repeat step 3.
5. Pour off fixing solution. Rinse gel and tray in ddH2O. Gently agitate for 5 minutes.
6. Remove gel, rinse container, and place gel in sufficient volume of 1X Krypton stain to cover gel. Cover the tray with foil and gentle agitate for at least 1 hour. Staining longer up to o/n might improve visualization of some proteins.
7. Remove gel, rinse container and place gel in a container with enough Destaining solution to cover gel. Cover the tray and agitate for 5 min.
8. Remove gel, rinse container and place gel in a container with enough ddH2O to cover gel. Cover the tray and agitate for 15 min.
9. Remove gel, rinse container and place gel in a container with enough ddH2O to cover gel. Cover the tray and agitate for 15 min.
10. Remove gel, rinse container and place gel in 1L stabilization solution and agitate gently for 30 min.

Coomassie Blue Stain
• Invitrogen’s iProtocol from Current Protocols in Molecular Biology
• Non specific binding of Coomassie brilliant blue R to proteins. The proteins are first ppt with a fixing solution, then the ppt proteins are stained with Coomassie blue.

Reagents and Solutions
Fixing solution
10% acetic acid, 50% methanol – 1L

100 ml glacial acetic acid
500 ml methanol
400 ml ddH2O

Staining solution
50% methanol/0.05% Coomassie brilliant blue R-250 (Bio-Rad)/ 10% acetic acid – 1L

Dissolve Coomassie brilliant blue R-250 in methanol before adding acetic acid and H2O. Store for up to 6 months at RT

100 ml glacial acetic acid
500 ml methanol
0.5 gm Coomassie brilliant blue R-250
400 ml ddH2O

Destaining solution
7% acetic acid, 5% methanol – 1L

70ml glacial acetic acid
50ml glacial acetic acid
880ml ddH20

Protocol
1. Place the gel in a plastic container and cover with 3 – 5 gel volumes of fixing solution. Agitate slowly 2 hrs. at RT on an orbital shaker or rocking platform.
2. Pour off fixing solution (do not reuse). Cover the gel with Coomassie blue staining solution for 4 hr and agitate slowly.
3. Pour off the staining solution (do not reuse). Rinse the gel briefly with ~50 ml fixing solution.
4. Pour off the fixing solution. Cover the gel with destaining solution for 2 hr. and agitate slowly.
5. Pour out destaining solution. Add fresh destaining solution and continue destaining until blue bands and a clear background are obtained.
6. Store the gel in 7% acetic acid or H2O.

Simply Blue SafeStain Invitrogen
• Fast and easy; destaining is not required
• Eliminates Reagent and solution prep
• Safe non-hazardous disposal

Basic Protocol
1. Rinse the gel 3 times for 5 minutes each with ddH2O. Discard each rinse.
2. Stain the gel with SimplyBlue SafeStain to cover gel. Stain for 1 hour at RT with gentle agitation. Discard the stain – it can not be reused. If you have to leave the gel o/n, add 2 ml of 20% NaCl for every 20 ml of stain.
3. Wash the gel with water to cover gel for 1 –3 hours. The gel can be left in water for several days.
4. For the clearest background, repeat step 3. However, now you need to add NaCl (see step 1) if storage is longer than 1 day.

Maximum Sensitivity
1. Prepare a 20% NaCl solution in ddH2O. You will need 20 – 30 ml/gel depending on the size of the gel.
2. Perform step 1 and 2 in Basic protocol.
3. After staining the gel, wash the gel with water to cover gel for 1 hour.
4. Add 20 (30) ml 20% NaCl to the water in step 3 and continue to wash for an additional 2 hours or o/n if needed.

GelCode Blue Stain Reagent Pierce
• Fast and easy; destaining is not required

Protocol
1. SDS –PAGE gels: Rinse the gel 3 times for 5 minutes each with ddH2O. Or wash gel in 1 –2 L of ddH2O with gentle shaking for 15 min.
2. Mix the GelCode Blue Stain Reagent solution immediately before use by gently inverting or tipping and swirling the bottle several times. Do not shake the bottle to mix the solution.
3. Add 20 ml of GelCode Blue Stain Reagent for a 8 X 10 cm gel. Gently shake the tray and watch band development. It should take about 1 hour. Gels can be stained o/n without increasing background.
4. Destain (optional). Replace Stain Reagent with ddH2O. Several water changes for a 1 –2 hour period may be necessary for optimal results. This step enhances stain sensitivity.

PROTSIL1 ProteoSilver Silver Stain Kit Sigma
• Premixed solutions reduces time and cost.
• High sensitivity and low background
• MALDI compatible
• Room temperature Stability

 


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




 

 

2d analysis 2D 1st dimension 2D 2nd dimension 2D imaging 2D scanners 2D imagemasetr 2D decyder 2d spotpicker 2D tables 2D trypsin 2D ziptip facscanto image-quant

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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