Cyanogen Bromide digestion of protein
From:putus.org Author:putus.org Time:3/2/2011 6:45:20 PM Count:
1. Proteins are TCA precipitated and washed with acetone, then dried.
2. The CNBr should be brought to room temperature in the hood and used ONLY in the hood. Extremely toxic!
3. Dissolve a small amount of CNBr in 70% TFA, the exact amount is not critical. One or two crystals should be fine, although weighing this reagent is NOT recommended. Place the utensil used in dilute bleach to neutralize the residual CNBr.
4. Redissolve the precipitated protein in 100 microliters of CNBr/TFA solution, close tube.
5. Digestion of the protein will take about 36 to 72 hours at room temperature.
6. Dry the samples by Speedvac with heat for an hour. Be very careful of lingering fumes when removing samples from the Speedvac.
7. Redissolve the digested protein in water or other buffer. These samples are somewhat acidic and should be neutralized with 1M Tris 8.0 before loading to SDS PAGE.
2. The CNBr should be brought to room temperature in the hood and used ONLY in the hood. Extremely toxic!
3. Dissolve a small amount of CNBr in 70% TFA, the exact amount is not critical. One or two crystals should be fine, although weighing this reagent is NOT recommended. Place the utensil used in dilute bleach to neutralize the residual CNBr.
4. Redissolve the precipitated protein in 100 microliters of CNBr/TFA solution, close tube.
5. Digestion of the protein will take about 36 to 72 hours at room temperature.
6. Dry the samples by Speedvac with heat for an hour. Be very careful of lingering fumes when removing samples from the Speedvac.
7. Redissolve the digested protein in water or other buffer. These samples are somewhat acidic and should be neutralized with 1M Tris 8.0 before loading to SDS PAGE.
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