Sauer:Silver staining: Difference between revisions
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== | =Protocol 1= | ||
The recommended volume of solutions for mini-gels is 50 mL | The recommended volume of solutions for mini-gels is 50 mL | ||
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[[Image:silver ge2.jpg]] | [[Image:silver ge2.jpg]] | ||
=Protocol 2= | |||
==Background== | |||
Silver staining is useful for detecting small amounts of proteins (2–5 ng/band). It is up to 200 times more sensitive than coomassie blue staining. Some commercial stains, like [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=1100&CMP=LEC-GCMSSEARCH&HQS=S21900 SYPRO Ruby protein gel stain] from Molecular Probes/Invitrogen, are much easier to use than silver staining and claim to have the same, if not better, sensitivity. | |||
==Procedure== | |||
This procedure involves incubation of your gel in different solutions. Use enough volume of each solution such that your gel(s) are completely covered with liquid and can shake freely in the liquid. Typically, 50 mL per minigel is sufficient. It is also very important that clean containers are used and that you wear gloves to prevent proteins from your hands from getting on the gel. All steps should be done at 4 ˚C. | |||
#Fix gel in 50% methanol, 5% acetic acid for 20 min. | |||
#Wash in 50% methanol, 10 min. | |||
#Wash in H<sub>2</sub>O, 10 min. | |||
#Sensitize in 0.02% sodium thiosulfate (Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>•5H<sub>2</sub>O; make fresh each time!), 1 min. | |||
#Rinse 2 times with H<sub>2</sub>O for 1 min each time. | |||
#Stain with 0.2% silver nitrate (AgNO<sub>3</sub>; make fresh each time!) for 20 min. | |||
#Rinse 2 times with H<sub>2</sub>O for 1 min each time. | |||
#Develop with 0.04% formaldehyde/2% sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>). Pay attention to this step and have the stop solution ready. You want to stop the reaction when you can see the bands on your gel. Unfortunately, the best way to tell when to stop the reaction is to screw it up a few times. Nothing substitutes for experience, so good luck! (It can take anywhere from <1 min to 30 min. It's often a good idea to have some kind of standard on the gel that you know will stain such that you can use that as an indicator that the staining procedure worked.) | |||
#Stop the reaction with 1% acetic acid. You can store the gel indefinitely at 4 ˚C in 1% acetic acid, but I would recommend taking a picture/image of the gel or drying it at this point. | |||
==Buffers== | |||
How to make the buffers should be self-explanatory. I usually make the sodium thiosulfate, silver nitrate, and formaldehyde/sodium carbonate solutions the day I am going to use them and keep the rest in bottles in the cold room. | |||
==Considerations== | |||
*Silver nitrate is light-sensitive. Wrap some aluminum foil around the solution when you make it up to keep it in the dark until you are ready to use it. | |||
*All solutions containing silver should be disposed of in the appropriate waste container, not down the sink. | |||
Latest revision as of 13:09, 21 January 2006
Protocol 1
The recommended volume of solutions for mini-gels is 50 mL
- 1hr (or more) 50% EtOH, 12%HOAc, 0.5 mL/L Formaldehyde (37%)
- 2 x 20min 50% EtOH
- 20 sec ddH2O
- 1 min 0.2 g/L Na2S2O3•5H2O
- 3 x 20 sec ddH2O
- 20 min 2 g/L AgNO3, 750 µL/L Formaldehyde (37%)
- 2 x 20 sec ddH2O
Prepare developing solutions in advance!
- 30 g/L K2CO3, 500 µL/L Formaldehyde (37%), 4 mg/L Na2S2O3.5H2O (for 50 mL just use the smallest granule you can find)
- Shake gently until developed (typically less than a minute)
- 1 min 1% Glycine
- 1 min ddH2O
- 10 min 1% Glycine
- 30 min ddH2O
- NOTE:Many of the silver stain solutions are hazardous waste and cannot go down the sink. Please dispose of them properly.
This is an example of how it should look:
Protocol 2
Background
Silver staining is useful for detecting small amounts of proteins (2–5 ng/band). It is up to 200 times more sensitive than coomassie blue staining. Some commercial stains, like SYPRO Ruby protein gel stain from Molecular Probes/Invitrogen, are much easier to use than silver staining and claim to have the same, if not better, sensitivity.
Procedure
This procedure involves incubation of your gel in different solutions. Use enough volume of each solution such that your gel(s) are completely covered with liquid and can shake freely in the liquid. Typically, 50 mL per minigel is sufficient. It is also very important that clean containers are used and that you wear gloves to prevent proteins from your hands from getting on the gel. All steps should be done at 4 ˚C.
- Fix gel in 50% methanol, 5% acetic acid for 20 min.
- Wash in 50% methanol, 10 min.
- Wash in H2O, 10 min.
- Sensitize in 0.02% sodium thiosulfate (Na2S2O3•5H2O; make fresh each time!), 1 min.
- Rinse 2 times with H2O for 1 min each time.
- Stain with 0.2% silver nitrate (AgNO3; make fresh each time!) for 20 min.
- Rinse 2 times with H2O for 1 min each time.
- Develop with 0.04% formaldehyde/2% sodium carbonate (Na2CO3). Pay attention to this step and have the stop solution ready. You want to stop the reaction when you can see the bands on your gel. Unfortunately, the best way to tell when to stop the reaction is to screw it up a few times. Nothing substitutes for experience, so good luck! (It can take anywhere from <1 min to 30 min. It's often a good idea to have some kind of standard on the gel that you know will stain such that you can use that as an indicator that the staining procedure worked.)
- Stop the reaction with 1% acetic acid. You can store the gel indefinitely at 4 ˚C in 1% acetic acid, but I would recommend taking a picture/image of the gel or drying it at this point.
Buffers
How to make the buffers should be self-explanatory. I usually make the sodium thiosulfate, silver nitrate, and formaldehyde/sodium carbonate solutions the day I am going to use them and keep the rest in bottles in the cold room.
Considerations
- Silver nitrate is light-sensitive. Wrap some aluminum foil around the solution when you make it up to keep it in the dark until you are ready to use it.
- All solutions containing silver should be disposed of in the appropriate waste container, not down the sink.
