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Silver staining (Jensen ‘99)
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This silver staining protocol is suitable for staining gel prior to analysis of the theproteins by peptide mapping by mass spectrometry.
After running the gel as usual proceed to the following fixation and staining steps while taking outmost care not to contaminate the gel with "finger proteins". Wash gloves before use. Use fresh sample buffer to reduce keratins.
Fixation 45% methanol and 5% acetic acid 30min Wash DDW 10 min. Wash DDW 60 min Sensitization 0.02% Sodium thiosulfate 1-2 min Wash DDW 2 x 1 min Staining Chilled 0.1% silver nitrate 30 min at 4C Wash DDW 2 x 1 min Develop 0.04% formaldehyde and 2% Na2CO3 with shaking. Change to fresh solution when turns yellow. Termination 5% acetic acid
All solution should be made fresh.
Note: The gels are cast together with 5mM sodium thiosulfate
The protocol is according to Jensen, O. N. et al (1999), sample preparation methods
for mass spectrometric peptide mapping directly from 2-DE gels. Methods in Molecular Biology.112:512-530
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Silver staining (Shevchenko et al)
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Comments
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All steps are performed on a shaking table at room temperature except step 5.
Fix gel with destaining solution
· methanol: acetic acid : water (40 : 10 : 50)
· time: 20 - 30 min
Rinse gel with water to remove acid
· several times
· time: 3 - 4 hours
Sensitize gel with 0.02% (w/v) sodium thiosulfate
· 0.1g sodium thiosulfate in 0.5L water
· time: 1 - 2 min
Rinse gel with two changes of water (1 min each)
Incubate gel in (chilled ) 0.1% (w/v) silver solution
· 0.5g silver nitrate in 0.5L water
· time: 20 - 40 min, incubate at 4°C
Rinse gel with two changes of water (1min each)
Develop gel with developing solution (0.04% (v/v) formaldehyde, 2% (w/v) sodium carbonate)
· 10g sodium carbonate in 0.5L water
· 540µl formaldehyde (37%) in 0.5L water
· on a shaking table
· replace solution when it turns yellow
Quench development when sufficient staining is obtained
· add 1% acetic acid
· 5 ml acetic acid in 0.5L water
Store the silver stained gel in 1% (v/v) acetic acid solution at 4°C
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this is the classical and many times approved protocol for silverstained proteinspots and followed by maldi-tof-ms / pmf
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Coomassie Brilliant Blue (CBB) staining
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Prior to Coomassie Blue staining, proteins are usually
-fixed for >30 min in 20% TCA.
-After a brief (5 min) washing step with 40% ethanol and 10% acetic acid,
the gel is saturated with the dye solution (0.1% Serva Blue R-250 in 40% ethanol and 10% acetic acid) for 3 hours, and destained (however not completely!) two times 20 min each with 40% ethanol and 10% acetic acid until the background is no longer stained dark blue. Then the gel is immersed in 500 ml of distilled water containing a few ml of acetic acid for up to 48 hours to decrease background staining and to increase sensitivity. Detection limit of this stain is better than one µg protein/spot.
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Colloidal Coomassie Blue Staining Procedure (cCBB-stain)
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- Fix gels in 40% methanol/10% Acetic Acid 30 minutes
- Rinse in deionized water 2 minutes
- Add Rothi Blue Colloidal and shake gently for 1-1.5 hrs covered. (Best if left overnight)
- Rinse/intensify with 10% Ethanol 2 x 30 minutes each.
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this staining can be drastically enhanced using sypro ruby before cbb stain.
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Fluorescent staining with SYPRO™Ruby according to Jackson
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1. Remove the gel from the electrophoretic cassette
2. Fix the proteins for 30 min ethanol/acetic acid/water (10:7:83)
2. Place the gel in a high density polypropylene box. Do NOT use a glass vessel!
3. Add 500 ml SYPRO Ruby stain solution for a gel with the dimensions 250mm x 200mm
Note: Up to three gels may be stained simultaneously in one polypropylene box
4. Cover box with tight-fitting lid and with Aluminium foil to protect the reagent from
bright light
6. Shake the box gently, preferably with a circular action, at least 3 h at room
temperature
7. Pour off the excess stain solution and discard
8. Place the gel on a Pyrex glass plate
9. Image the gel using an appropriate fluorescence imager
Note: Use the filter sets that match the excitation and emission wavelength for SYPRO
Ruby. Images can also be obtained by using a simple UV transilluminator. However good sensitivity and resolution will only be obtained with dedicated imagers. Best is to destain in steps of 10 min with 10% ethanol and 7% acetic acid until you get an optimal image.
Additional information
Fixing the gel is not absolutely necessary
The stain should not be diluted or re-used in order to avoid loss of sensitivity
Destaining is not always required, but the background can be decreased by washing the gel with water or in ethanol/acetic acid/ water (10:7:83), for 30-60min. This procedure will also reduce speckling which can be a problem
The gels should be viewed on a Pyrex glass plate. Other glass plates have significant intrinsic fluorescence that will reduce image clarity and sensitivity
There is no correct exposure time which is valid for all experiments; each investigator will need to use a time suited to their requirements for a particular sample
When using gels that have a plastic backing it is advisable to place the gel with the gel-side down onto the UV transilluminator
SYPRO Ruby does not stain nucleic acids
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Silver staining protocol according to Oakely et al. (1980) (modified)
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Use reagent-grade chemicals! The conductivity of the deionized or distilled water should not exceed 2 µS!
Reagent solutions
Fixative:
40 % Ethanol, 10% acetic acid, 50% deionized water
Glutardialdehyde solution: 6.25% glutardialdehyde
Glutardialdehyde (25%) 250 ml
Deionized water fill up to 1000 ml
Silver reagent:
0.2% silver nitrate in 0.25% ammonia, 5% sodium hydroxide (1M), 20 % ethanol
Dissolve 2 g silver nitrate in 10 ml of deionized water; add 10 ml ammonia (25%), 50 ml sodium hydroxide (1M), 730 ml deionized water and 200 ml ethanol.
Developer:
0.1% formaldehyde (37%), 0.025% citric acid (2.3 M), 20% ethanol
Formaldehyde (37%) 1 ml
Citric acid (2.3 M) 0.25 ml
Ethanol (20%) 1000 ml
Stop-reagent: 1% glycine
Glycine 10 g
Deionized water fill up to 1000 ml
Fixing 40% methanol / 10% acetic acid 30 min Wash deionized water 2 x 5 min Sensitizing Glutardialdehyde solution 20 min Wash 20% ethanol 15 min Wash deionized water 2 x 10 min Silver Silver reagent 30 min Wash 20% ethanol 2 x 5 min Development Developer 1 x 30sec, 1 x 5 min Wash deionized water 1 x 20 sec Stop Stop reagent 5 min Wash deionized water 3 x 10 min
Do not touch the gel with your fingers. Always wear gloves or use forceps.
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Silver staining protocol according to Heukeshoven and Dernick (mod.)
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Use reagent-grade chemicals! The conductivity of the deionized or distilled water should not exceed 2 µS!
Reagent solutions
Fixative:
40 % Ethanol, 10% acetic acid, 50% deionized water
Sensitizer:
0.3% sodium thiosulfate, 0.5% glutardialdehyde, 30% ethanol in sodiumacetate-buffer
Sodium acetate 68g
Sodium thiosulfate pentahydrate 3 g
Glutardialdehyde (25%) 20 ml
Deionized water 700 ml
Ethanol 300 ml
Silver nitrate reagent:
0.1% silver nitrate, 0.05% Formaldehyde (37%)
Silver nitrate 1 g
Formaldehyde (37%) 0.5 ml
Deionized water fill up to 1000 ml
Developer:
3% sodium carbonate, 0.025% formaldehyde (37%)
Sodium carbonate 30 g
Formaldehyde (37%) 0.25 ml
Deionized water fill up to 1000 ml
Stop-reagent:
1% glycine
Glycine 10 g
Deionized water fill up to 1000 ml
Step Reagent & Duration
- Fixing: 40% methanol / 10% acetic acid 30 min
- Wash: deionized water 5 min
- Sensitizing: Sensitizer 60 min
- Wash: deionized water 6 x 10 min
- Silver: Silver nitrate reagent 30 min
- Wash: deionized water 3 x 20 sec
- Development: Developer 3-5 min
- Wash: deionized water 1 x 20 sec
- Stop: Stop reagent 5 min
- Wash: deionized water 3 x 10 min
Do not touch the gel with your fingers. Always wear gloves or use forceps.
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this staining procedure is the most sensitive silverstain up to now. unfortunately it is not compatible with maldi-tof-ms but the best one for documentation reasons
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Silver staing protocol according to Blum et al. (1987) (modified)
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Use reagent-grade chemicals! The conductivity of the deionized or distilled water
should not exceed 2 µS!
Reagent solutions
Fixative:
40 % Ethanol, 10% acetic acid, 50% deionized water
Wash-solution:
30% Ethanol, 70% deionized water
Thiosulfate reagent: 0.02% sodium thiosulfate
Sodium thiosulfate 200 mg
Deionized water fill up to 1000 ml
Silver nitrate reagent: 0.2% silver nitrate, 0.02% Formaldehyde (37%)
Silver nitrate 2 g
Formaldehyde (37%) 0.2 ml
Deionized water fill up to 1000 ml
Developer: 3% sodium carbonate, 0.05% formaldehyde, 0.0005% sodium thiosulfate
Sodium carbonate 30 g
Formaldehyde (37%) 0.5 ml
Sodium thiosulfate 5 mg
Deionized water fill up to 1000 ml
Stop-reagent: 0.5% glycine
Glycine 5 g
Deionized water fill up to 1000 ml
Step Reagent Duration
- Fixing: 40% methanol / 10% acetic acid > 1 h
- Wash: 30% ethanol 2 x 20 min
- Wash: distilled water 1 x 20 min
- Sensitizer: Thiosulfate reagent 1 min
- Wash: deionized water 3 x 20 sec
- Silver: Silver nitrate 20 min
- Wash: deionized water 3 x 20 sec
- Development: Developer 3-5 min
- Wash: deionized water 1 x 20 sec
- Stop: Stop reagent 5 min
- Wash: deionized water 3 x 10 min
Do not touch the gel with your fingers. Always wear gloves or use forceps.
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the classical silverstaining protocol
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Lightning Fast / Deep Purple staining protocol V1.6
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- Fix: Place the gel in a solution of 7.5 % acetic acid and transfer to a rocker or orbital shaking platform for 1 hour. It is recommended that the volume of fixative is approximately 10 X the gel volume. For example, use a 500 mL volume for a large format gel (20 cm x 18 cm x 1.5 mm) or 100 mL for a mini gel (10 cm x 10 cm x 1.0 mm). The use of clean polypropylene containers is recommended for the gel staining steps.
- Wash: Discard all of the acetic acid from the staining box and wash the gel 2 x 30 minutes in 10 X gel volumes of double distilled (dd) water.
- Stain: Place the gel in fresh dd water and add Lightning Fast. Lightning Fast is supplied as a 200 X concentrate. Recommended staining volumes are 2.5 mL of Lightning Fast per 500 mL of dd water to stain a large format gel. For mini gels the recommended volumes are 250 µL of Lightning Fast in 50 mL of dd water. Place the gel box on a rocking platform and stain for 1 h. For optimal results use a dark staining box or wrap the gel box with foil to exclude light.
- Enhance: Remove all of the gel stain and wash the gel for 2 x 10 minutes in 10 X gel volumes of 0.05 % (v/v) (8 mM) ammonia solution. Remove all of the ammonia solution and add a 10 X gel volume of 0.5 % (80 mM) acetic acid to the gel and wash for a further 10 minutes. The gel is then ready for imaging. For longer term the gel should be stored in 0.5% (80 mM) acetic acid at 4o C.
- Image: For ultra high sensitivity detection it is recommended that fluorescent imaging systems such as the Amersham Typhoon or Bio-Rad FX are used. The use of a 532-nm YAG laser and 560 nm Long Pass or 610 Band Pass emission filter provide optimal results. CCD cameras used in conjunction with UVA or UVB transilluminators and emission filtering also provides high sensitivity detection. Proteins can be identified by the human eye using UVA, UVB or blue light transilluminators in conjunction with orange glasses. Lightning Fast can be removed from the glass surface of imaging devices using mild detergent such as Pyroneg. For optimal results ensure the imaging platen is clean before use.
Do not touch the gel with your fingers. Always wear gloves or use forceps.
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