Protein Staining Procedures

n diH2O for 1 hr. Gels can be stored at 4°C. Silver Nitrate Staining A: Solutions: 1. Ammonical silver nitrate staining solution:Prepare solutions A and B as described below and, after mixing together, make to the final volume. Dissolve each solution WELL by vortexing vigorously. Solution A Solution B diH2O (ml) 10 N NaOH (ml) NH4OH (ml) Silver Nitrate (g) diH2O (ml) Final Volume (ml) 21 0.2 1.3 1.5 4 100 31.5 0.3 1.95 2.25 6 150 105 1 6.5 7.5 20 500 210 2 13 15 40 1000 315 3 19.5 22.5 60 1500 420 4 26 30 80 2000 Add solution B to solution A slowly. A brown precipitate will appear then disappear. If disappearance of the brown precipitate is slow, add drops of NH4OH. Bring to final volume.TIP: diH2O should be high quality. Otherwise, silver nitrate will precipitate. 2. Developing solution ( 0.1% formaldehyde, 0.01% citric acid): 100 µl formaldehyde and 0.01 g of citric acid /100 ml diH2O3. Stop solution (2% acetic acid): 2 ml acetic acid plus 98 ml diH2O4. Destaining solution: Prepare separately: 10 mM sodium thiosulfate pentahydrate ( 0.248 g/100 ml diH2O) 30 mM potassium ferricyanide (0.988 g/100 ml diH2O) Mix both solutions together. B: Procedure:All washes and incubations are done with constant, gentle shaking. 1. StainingCut gels at the top/basic corner. Immerse in 50% ethanol/10% acetic acid for at least 1 hr. Soak in 5% ethanol/5% acetic acid overnight for a minimum of 2 hours to several days. Wash in diH2O for 1 hr. Fix in 1% glutaraldhyde/0.5 M sodium acetate for 30-45 min. Wash in diH2O, three times for 15 min each. Add Gel-Code Blue Stain reagent (Pierce, #24592) for at least 3 hrs. Wash in diH2O twice for 15 min each. Wash in diH2O for 1 hr. Stain gel in ammonical silver nitrate solution for 1 hr. Rinse gel three times for 5 min each in diH2O. Develop gel in developing solution for 5-10 min or until signal to noise is appropriate. Stop development by adding 100-150 ml of the stop solution. To re-stain, rinse gel in diH2O three times for 15 min each, then repeat starting from the incubation with silver nitrate. 2. DestainingIf the gel is over-developed, it can be destained by adding the destaining solution to the gel for 30 sec-5 min. The silver staining should fade away slowly. At the right moment, scan the gel and save. Otherwise, rinse extensively in diH2O five or six times. Repeat silver staining. Note: Staining intensity may be less than normal. Zinc Imidazole Negative Staining A: Solutions:1. 1% sodium carbonate: 1 g sodium carbonate/100 ml diH2O 2. 200 mM imidazole/0.1% SDS: 1.36 g imidazole and 0.1 g SDS per 100 ml diH2O 3. 100 mM zinc acetate: 2.2 g zinc acetate/100 ml diH2O, filter through a 0.45 µm filter. B: Procedure: All washes and incubations are done with constant, gentle shaking.Rinse gels in HPLC grade H2O for 5 min. Equilibrate gel in 1% sodium carbonate for 15 min. Incubate gels in imidazole/SDS solution for 30 min. Rinse gels in HPLC grade H2O for 1 min. Develop gels in zinc acetate solution for 1-5 min or until spots are well resolved. Rinse gels in HPLC grade H2O three times for 5 min each. Spots can be cut out of the gels for sequencing, or Gels can be: stored at 4°C up to one week without significant protein diffusion destained with 50 mM EDTA and washed for: re-staining protein transfer silver staining
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