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Edman Degradation Sample Preparation Protocols : removal of salts and detergents

자료는 Edman Degradation 을 기초로 하는 sequencing sample 을 준비하는 과정에서 필요로 하는 전처리 과정의 방법을 자세히 기술한 자료입니다.

Protocol for Chloroform Methanol Precipitation
For Removal of salt and detergents
(본 방법은 시료내에 salt 나 detergent 가 포함되어져 있을 때, 제거하기 위한 유용한 방법입니다.)

1. To sample of starting volume 100 ul
2. Add 400 ul methanol
3. Vortex well
4. Add 100 ul chloroform
5. Vortex
6. Add 300 ul H2O
7. Vortex
8. Spin 1 minute @ 14,0000 g
9. Remove top aqueous layer (protein is between layers)
10. Add 400 ul methanol
11. Vortex
12. Spin 2 minutes @ 14,000 g
13. Remove as much MeOH as possible without disturbing pellet
14. Speed-Vac to dryness
15. Bring up in 2X sample buffer for PAGE

Reference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143

Protocol for Concentrating Proteins Prior to SDS-PAGE

Protein samples containing Guanidine-HCl (up to 4 µl of 8 M Guanidine-HCl):
1. Add 5% N-Lauroyl sarcosine (Sigma L-5125 C15 H28 NO3 Na, FW 293.4) to prevent guanidine from precipitating to 2x SDS Sample Buffer containing 10 mM DTT. Reduce and alkylate sample.

Microconcentration of proteins to remove salts or unwanted contaminants:
1. Choose microconcentrator tube with a protein cutoff smaller than molecular weight of the protein in sample. Amicon Microcon-10 MW Cutoff 10000 Amicon Centricon-30 MW Cutoff 30000
2. Add 1 µl of 20% w/v SDS to dry microcon tube (if sample does not already contain SDS).
3. Slowly add sample (a few microliters at a time) to membrane until membrane is completely wet. Centrifuge to near (but not complete) dryness.
4. If intention is to desalt sample or buffer exchange: Add ~50 µl water to microcon and spin until nearly dry. Repeat buffer exchange. Sample will remain on membrane.
5. Use new collection tube. Invert membrane and spin at low speed (1000x g) to elute protein from membrane. Add 2x SDS-Sample Buffer containing 10 mM DTT to membrane: vortex, invert and spin. Final volume should be ~20 µl. Reduce and alkylate sample according to standard protocol.

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