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[Proteomics] Process stained polyacrylamide gel pieces for mass spectrometry -
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본 실험 과정은 1D 또는 2D PAGE 를 수행하여 얻어진 단백질의 동정을 위한 실험을 수행하기 위하여 앞서 본 시료에 전 처리를 하는 과정을 설명하기 위한 내용으로 구성 되어져 있습니다.
본 과정은 coomassie blue 염색이 된 시료에 대하여 최종 mass 분석 전까지의 과정을 기술하기로 합니다.
추가적인 시료 (silver staining 시료) 의 경우에는 본 내용의 출처자료의 원문을 참고하시기 바랍니다.
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Gel electrophoresis of protein samples, trypsin digestion of target proteins and analysis of the resulting peptide fragments by mass spectrometry (MS) comprise a powerful method for protein identification and characterization. A fluorescent, coomassie or silver stain is necessary to visualize proteins that have been separated in 1-D or 2-D gels. Processing such samples for mass spectrometry necessitates first excising the protein spot of interest, removing the stain, and digesting and eluting the protein in the gel piece using an in-gel tryptic digestion procedure.
Procedure for Gels Stained with Coomassie
Note: The following procedure may be used for coomassie or fluorescent dye-stained polyacrylamide gel pieces. If not certain of the quality of the available reagents and trypsin, use the In-Gel Tryptic Digestion Kit
Material Preparation
Destaining Solution: 25 mM ammonium bicarbonate in 50% acetonitrile. Mix 80 mg of ammonium bicarbonate with 20 ml of acetonitrile and 20 ml of ultrapure water. Store this solution at 4°C for up to 2 months.
Digestion Buffer: 25 mM ammonium bicarbonate in water. Mix 10 mg of ammonium bicarbonate with 5 ml of ultrapure water. Store Digestion Buffer at 4°C for up to 2 months.
A. Excise and Destain Gel Piece
1. Use a spot picker or scalpel to excise protein band of interest from 1-D or 2-D gel. Cut band into 1×1 to 2×2 mm pieces. Place pieces into a receiver tube.
2. Add 200 ul Destaining Solution to gel pieces. Incubate sample at 37°C for 30 minutes with shaking.
3. Remove and discard Destaining Solution from the tube.
4. Repeat steps A.2-A.3.
5. Proceed to step B.1.
Note: Reduction and alkylation of the protein sample are optional but recommended if high-sequence coverage is desired. Refer to instructions for the In-Gel Tryptic Digestion Kit for a detailed protocol for reduction and alkylation.
B. Shrink Gel Pieces
1. Shrink gel pieces by adding 50 μl of acetonitrile. Incubate sample for 15 minutes at room temperature.
2. Carefully remove acetonitrile and allow gel pieces to air-dry for 5-10 minutes.
C. Trypsinize Proteins and Recover Fragments
1. Prepare a 1 μg/0.1 ml solution of high-quality trypsin in ultrapure water. Do not attempt to store this solution.
2. Add 10 μl of preprared trypsin solution to the tube containing the shrunken gel pieces; incubate at room temperature for 15 minutes to allow gel pieces to swell and absorb the trypsin solution.
Notes:
a) Using 100 ng of trypsin per digest is effective for a wide variety of protein concentrations within an excised gel band. However, if protein band contains significantly less than ~20 ng protein (~300 fmol), 25 ng of trypsin may beused per digest.
b) If 10 μl is not sufficient to cover and fully swell gel pieces, increase volume of trypsin solution accordingly.3. Add 25 μl of Digestion Buffer to the tube. Incubate sample at 37°C for 4 hours or at 30°C overnight with shaking.
4. Remove digestion mixture and place in a clean tube.
5. (Optional) To further extract peptides, add 10 μl 1% trifluoroacetic acid or 1% formic acid solution to gel pieces and incubate for 5 minutes. Remove extraction solution and add to digestion mixture (step 6). This step also serves to inactivate trypsin, stopping additional enzymatic activity. A second extraction generally results in only a minor increase in peptide recovery.
6. Sample is now ready for liquid chromatographic separation and electrospray ionization mass spectrometry (LC-ESI MS). Additional processing/clean-up by C-18 resin (Product No. 89870) is required for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or nanospray ionization mass spectrometry.
Note: To prevent clogging or column damage, ensure sample is free of any acrylamide pieces before applying to a LC/ESI/MC system.
자료 출처 : PIERCE technical resource