Portuguese Proteomics Network
Rede Proteómica Portuguesa
Protocols from Procura Members
Cell lysis and sample preparation:
– CHO cells
– MEL cells
– Silver staining protocol
– Silver staining protocol for MS
– Coomassie Brilliant Blue R-250
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Cell lysis and sample preparation
CHO cells were lysed in 350µl of lysis buffer supplemented with a cocktail of protease inhibitors. After 1h of incubation at RT, samples were clarified by centrifugation at 12.000 g for 5min.
Murine ErythroLeukemia (MEL) cells
MEL cells (1,5-2×106) were lysed in 100µl of lysis buffer containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM), tiurea (2 M) and TBP (2 mM). After that, the cells were rehidratated with 250 µl of rehidratation buffer composed by urea (8 M), CHAPS (2% w/v), DTE (10 mM), anpholites (pH 3,5-10, 2 % v/v) and some bromophenol blue grains. Before rehidratation MEL cells were left at 33ºC for 4 hours.
The samples were loaded onto an 18cm non-linear wide-range immobilized pH 3-10 gradient strip (Pharmacia’s pH 3.5-10 NL strip). After an active re-hydration (12h at 30V) at 20ºC, IEF was completed using a focusing programme in which the voltage is gradually increased up to 5000 V.
To resolubilize proteins, strips were soaked for 15min in equilibrating solution, then blocked for another 15min in blocking solution to reduce -S-S- bonds and finally applied to a 7-16% (w/v) gradient polyacrylamide gel. The second dimension runs at 80mA for 7h in denaturing conditions (SDS).
Silver staining protocol
All steps were performed on an orbital shaker at 36 rpm.
– When the second dimension was finished, the gel was removed from the glass plates and washed in deionized water for 5 min.
– After that the gel was fixed in ethanol (40 % v/v) and acetic acid (10 % v/v) for 1 hour.
– Again it was fixed in ethanol (5 % v/v) and acetic acid (5 % v/v) for 2 hours or overnight.
– Once more gel was washed in deionized water for 15 min.
– Later it was fixed in a solution of glutaraldehyde (1% v/v) and sodium acetate (0,5 M) for 30 min.
– Then gel was washed 3 times in deionized water for 10 min.
– During 30 min gel was stained in a freshly made ammoniacal silver nitrate solution. The preparation of 750 ml of this solution consists in dissolving 6 g of silver nitrate in 30 ml of deionized water, which was slowly mixed into a solution containing 160 ml of water, 10 ml of concentrated ammonia (25 % v/v) and 1,5 ml of sodium hydroxide (10 N). A transient brown precipitate might form. After it cleared, water was added to give the final volume.
– After staining process, the gel was washed 4 times in deionized water for 4 min.
– For 5 to 10 min the image was developed in a solution containing citric acid (0,01 % w/v) and formaldehyde (0,1 % v/v).
– Development was stopped with a solution containing Tris (5 % w/v) and acetic acid (2 % v/v) when a slight background stain appeared.
Silver staining protocol for MS
– After the gels’ run, it was fixed for 20 to 30 min in a fix solution.
– Then, it was rinsed in water for 30 to 60 min. To improve the contrast of the stained gel, longer incubation time is needed (e.g. 12 h).
– With 0,02 % sodium thiosulfate for 1 to 2 min, the gel was sensitized (glutaraldehyde should not be used because it is a protein crosslinking agent).
– The solution was removed and the gel slab was rinsed with two changes of water (1 min each).
– After that the gel was incubated in chilled 0,1 % AgNO3 solution for 20 to 40 min at 4ºC (in refrigerator).
– Once again the solution was removed and the gel slab was rinsed with two changes of water (1 min each).
– The gel was developed with a solution of 0,04 % formaldehyde/2 % Na2CO3 solution on a shaking table. It’s important to replace the developing solution when it turns yellow. Do not overexpose the gel.
– The developer solution was discarded and 1 % acetic acid was added when sufficient staining were obtained (usually after 0,5-5 min).
– In the end the silver stained gel was stored in 1 % acetic acid or in water at 4ºC.
Coomassie Brilliant Blue R-250
– At the end of the second dimension run, the gel was stained in a solution containing Coomassie Brilliant Blue R-250 (0,1 % w/v) and methanol (50 % v/v) for 15 min. Then the gel was destaining in a solution containing methanol (40 % v/v) and acetic acid (10 % v/v).