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[Proteomics] Protein Precipitation Methods -
작성자
이매스 -
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A. Acetone/ TCA Precipitation – works best for dilute samples (특히, 단백질 LC/MS 분석에 적합한 방법)
Chemical reagents (Reagents should be HPLC or USP grade):
• Trichloroacetic acid (TCA)
• Acetone
Procedure:
1. Mix 10 volumes of cold 10% TCA in Acetone (stored –20oC) with your samples, vortex, and incubate at –20oC for at least 3hrs, but overnight is optimal.
2. Centrifuge samples at 15000xg X 10min and remove supernatant.
3. Add the same volume of cold Acetone only, vortex, and let stand at –20oC for at least 10min.
4. Centrifuge at 15000xg X 5min, remove supernatant and allow pellets to air dry. Care should be taken to prevent complete desiccation of the protein pellet as this will make re-solubilization much harder.
Protein Re-solubilization
The protein precipitates need to be re-solubilized in a buffer compatible with the next step of analysis. For example: SDS-PAGE sample buffer for 1D gel analysis, re-hydration buffer for 2D-gel analysis, or a small volume of fresh buffered 6M urea then diluted to an appropriate concentration for an in-solution digests and protein quantification.
B. Acetone precipitation of proteins
Chemical reagents (Reagents should be HPLC or USP grade):
• Cold (-20°C) acetone, a volume four times that of the protein samples to be precipitated
• Centrifuge tube, made of acetone-compatible material such as polypropylene and able to hold five times the sample volume.
• Centrifuge and rotor for the tubes used, minimum 13,000 x g required.
Protocol
1. Cool the required volume of acetone to -20°C.
2. Place protein sample in acetone-compatible tube.
3. Add four times the sample volume of cold (-20°C) acetone to the tube.
4. Vortex tube and incubate for 60 minutes at -20°C.
5. Centrifuge 10 minutes at 13,000-15,000 x g.
6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
Optional: If additional cycles of precipitation are necessary to completely remove the interfering substance, then repeat steps 2-5 before proceeding to step 7.
7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it may not dissolve properly.
8. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protein pellet.
C. Chloroform / Methanol Precipitation
Protocol
1. Add 4 volumes of methanol and vortex well.
2. Add 1 volume of chloroform and vortex.
3. Add 3 volumes of dH2O and vortex
4. Centrifuge for 2 min at 15 000xg – the proteins should be at the liquid interface
5. Remove the aqueous top layer and add 4 volumes of methanol – vortex.
6. Centrifuge for 2 min at 15 000xg
7. Remove as much liquid as possible without disturbing precipitate.
8. Speed-Vac sample to dryness or dry under nitrogen.
D. Precipitation with Ammonium Sulfate ( 단백질이 불활성화 되지 않는 방법 - 또 다른 정제과정 및 function study 가능)
Protocol
1. Take sample in a beaker containing a stir bar and place on magnetic stirrer
2. While sample is stirring, slowly add ammonium sulfate of a desired saturation level (Can refer ammonium sulphate precipitation chart)
3. Add ammonium sulfate very slowly to ensure that local concentration around the site of addition does not exceed the desired salt concentration.
4. Once total volume of ammonium sulfate is added, move beaker to 4°C for 6 hours or overnight.
5. Collect the precipitate from the beaker and centrifuge the precipitate at 5000g for 20 minutes.
6. Carefully remove and discard supernatant. Invert the tube and drain well
7. Give two or three wash with distilled water
8. Dissolve the precipitate in phosphate buffer saline and dialyze protein solution at low temperature overnight to get removed salt
9. Determine the concentration and store at -10°C for long term storage.
E. Polyethylene Glycol Precipitation( 단백질이 불활성화 되지 않는 방법 - 또 다른 정제과정 및 function study 가능)
Chemical reagents:
USP grade salts, Tween 20, hydrochloric acid, acetic acid, and sodium hydroxide were purchased from JT Baker (Phillipsburg, NJ). PEG was of reagent grade and purchased from JT Baker or EMD Chemicals (Gibbstown, NJ). All buffers were prepared using MilliQ-grade water (Millipore, Billerica, MA) and were filtered by 0.22-µm filtration before use.
Feedstock
A human MAb (IgG1, pI = 8.3, 150 kDa) was produced at Percivia, LLC using a PER.C6 cell line. PER.C6 cells are human embryonic retinal cells immortalized by the adenovirus E1 gene, as described in US patent 5,994,128.12 The cells were cultured in a standard fed-batch process or the XD process, both using chemically defined media.13,14 The fed-batch media were clarified by sedimentation and depth filtration, and the XD media were clarified by the enhanced cell settling (ECS) method followed by depth filtration.15 During ECS, Silica-PEI resin was used to enhance cell settling and also reduce DNA and HCP.
Precipitation Condition Optimization
The conditions used to precipitate the MAb—PEG molecular weight, PEG concentration, and pH—were optimized by full factorial experimental designs using Minitab software (State College, PA). The pH of the clarified XD media was adjusted to the desired level with 2-M Tris in a 15-mL conical tube. The PEG was added as a 40% (w/w) stock solution to the desired final concentration. The tube was then centrifuged at 1,000g and the supernatant decanted. Finally, the pellet was redissolved in phosphate-buffered saline (PBS).
Cation Exchange Chromatography
Toyopearl GigaCap S-650 was procured from Tosoh Bioscience (Montgomeryville, PA) in the Toyoscreen 5-mL format. This resin has been previously demonstrated as a high capacity capture step for MAbs.16 The column was equilibrated with 74-mM sodium acetate pH 5.3 and loaded to 90–95 mg-MAb/mL-resin using either clarified media or clarified and PEG-treated material, each adjusted to the same pH and conductivity as the equilibration buffer. The column was then washed with equilibration buffer and the antibody eluted with 50 mM sodium acetate pH 5.3 plus 90 mM NaCl.
References
Acetone precipitation of proteins., TECHNICAL RESOURCE., PIERCE
Protein Precipitation Procedures.,BMSL
Protein Purification Techniques., Technical Bulletin., SAFC biosciences
Ammonium Sulfate Protocol., DragonTech