In proteomics research, a key step in all enrichment techniques is the subcellular fractionation, which is particularly important for the analysis of intracellular organelles and multiprotein complexes. In order to reduce sample complexity, subcellular fractionation should be performed using a flexible and operational method and is most effective when combined with high-resolution 2D gel/mass spectrometry or stand-alone gel techniques.
Authors: Burgess et al., Translated by Chen Wei, this experiment is from "Protein Purification Guide".
Operation method
Experiments on the isolation of subcellular organelles and subcellular structures Move I. Extraction and preliminary isolation of sub-proteomes 1. Enrichment of mitochondria Materials and equipment (2) Protease inhibitor mixture. (3) Phosphate buffer solution (PBS): 1. 4 m m o l l / L KH2PO4 , 8 m m o l l / L Na2 HPO4, 140 m m o l l / L NaCl, 2.7 m m o l l / L KCl, pH 7. 3. (4) 200 mmol/L n-dodecyl-sheet 1> maltoside [10% (m/V) lauryl maltoside]. (5) Horizontal ultracentrifuge and rotor (swing-out ultracentrifuge and rotor). (6) Heterogeneous homocrystalline polymer centrifuge tubes. Cells are lysed and homogenized as described above. Mechanical fragmentation is preferred (Goldberg, 2008), avoiding the addition of detergents, which may lead to leakage of mitochondria and other cellular compartments. General protocol (1) Wash the cells twice with PBS. (2) Resuspend cells with PBSC l m L/109 cells) containing phosphatase inhibitor and protease inhibitor. Cells were crushed more than 200 times with a Potter-Elvehjem type or Dounce homogenizer (tested by active staining). (3) Cells may optionally be processed by 5 or more freeze-thaw cycles, 5 or more ultrasounds (5 s per ultrasound, 5 s pause), Fourier cell crusher, high-pressure homogenization (500 to 1000 bar, 2 cycles), or glass bead mill according to the protocols supplied by the respective manufacturers. For the next step, the cells were centrifuged at 500 g at 4°C. The cells were then processed in a single step. Steps (1) Add lauryl maltoside to a P B S suspension of mitochondrial membranes with a maximum protein concentration of 5 m g /m L to give a final concentration of 1 %. (2) Mix well and incubate in an ice bath for 30 m i n . (3) Centrifuge at 70 000 g for 30 m i n . (4) Collect the supernatant and discard the precipitate. (5) Add the protease inhibitor mixture to the sample and place it in an ice bath until the centrifugation operation begins. (6) If the sucrose gradient preparation is complete, a discontinuous distribution of sucrose layers is observed. Carefully add the sample to the top layer of the gradient in the centrifuge tube, keeping the tube stable during operation. (7) Place the heterogeneous homocrystalline polymer centrifuge tube in a larger bucket-type rotor set to the lowest acceleration and deceleration, and centrifuge for 16 h at 130,000 g at 4°C. The tube is then centrifuged to the top of the gradient. (8) Carefully remove 500 of the fractions from top to bottom for analysis. The remaining fractions are placed in a freezer at 80°C. The fractions are then frozen. Preparation of other density gradients For large amounts of proteins, correspondingly multiple gradients can be prepared or the previous gradient can be scaled up. For example, 40 m g of mitochondria can be separated by a sucrose gradient solution with a total volume of 40 m L. The gradient can also be scaled up. (1) A sucrose solution is used as follows: 55% (5 m L ), 50% (5 m L ), 35% (10 m L ), 30% (10 m L ) and 25% (2 m L ). (2) Add 8 t n L of mitochondrial supernatant (dissolved in 1 % lauryl maltoside and centrifuged at 72 000 g for 30 m i n ) to the top of the gradient solution. (3) Set the acceleration and deceleration to minimum and centrifuge for 17 h at 115 000 g at 4°C. In addition to the classical sucrose density gradient media, different media from other suppliers can also be used to isolate subcellular structures and organelles. The most prominent of these media are sucrose (Ficoll) (Ku r o k a w a etal., 1965), Percoll (Pertoft et al., 1977), Nycoden z (Rick w ood et al., 1982) and Optiprep (van Veldh o v e n e t a l ., 1996). , 1996). More detailed programs can be found in the operating manuals provided by the respective suppliers. Materials and Equipment (1) Homogenizing buffer :250 mmol/L sucrose. 10 mmol/L HEPES^NaOH(pH 7.4),1 mmol/LEDTACpH 8.0),I mmol/L DTT (2) Optiprcp storage solution: 60% iodixanol in water. (3) System buffer: 60 mmol/L HEPES-NaOH (p H 7.4), 6 mmol/L EDTA (p H8.0), 6 mmol/L DTT (4) Iodixanol Buffer: Prepared by adding I volume of system buffer to 5 volumes of Optiprep stock solution. Note: All solutions should be made up as they are used and stored at 4°C until used. Procedure (1) Pre-clarify the homogenate by centrifugation at 2200 g for 5 min at 4°C to remove intact cells, nuclei and cell wall fragments. (2) Gently pour the supernatant into a clean centrifuge tube and centrifuge again under the conditions of the previous step. (3) Divide the resulting supernatant into 6 equal portions and transfer to 6 centrifuge tubes, then add 6 mL1 of 8 % iodixanol buffer to the bottom of the tube. (4) Centrifuge at 100 OOOg for 2 h at 4°C using an ultracentrifuge. (5) Collect the crude extract of cellular biofilm from the intermediate phase (usually 6~8 mL). (6) The cell membrane extract was diluted using iodixanol buffer solution and the concentration of iodixanol was adjusted to 16 % using iodixanol buffer solution stock solution. (7) The adjusted solution was poured into 2 polycarbonate centrifuge tubes and centrifuged at 4°C for 3 h at 350 OOOg using an ultracentrifuge with the deceleration rate set to minimum. (8) Collect 2 0 0.5 mL of density fraction from the top of each gradient in the polycarbonate centrifuge tubes. (9) Add 800 pL of 162. 5 mmol/L Na2C a to each of the selected 0. 5 mL of density fractions to achieve a final concentration of 100 mmol/L. (10) Incubate in an ice bath for 30 min, centrifuge at 100,000 g for 25 min at 4°C using an ultracentrifuge, and discard the supernatant. (11) Wash the precipitate with I mL of deionized water at 4°C, centrifuge as above for 10 min, and discard the supernatant. Reagents and equipment (1) 100 m m o l / L E D T A : Weigh 3. 36 g of E D T A and dissolve in 100 mL of water. Store at room temperature. (2) 100 m m ol/L PMSF: Weigh 174 m g of PM SF and dissolve in 100 mL of isopropanol. (3) Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) storage buffer (10x ): Filtered through a 0.4 5um sterile filter and stored at 4°C, protected from light. (4) Cytosolic Protein Extraction Buffer (0.015% Digitalis Saponin, pH 6.8, 4°C): Take a flask with a small stirring bar and add 4 mL of IO X Storage Buffer, then dissolve 18.75 m g of Digitalis Saponin (calbi 〇 chem) in it by heating and then add I m L of PMSF. add I m L of PMSF to the remaining 6 mL of IO X Storage Buffer and 5 mL of PMSF. 5 mL (5) Cell membrane protein extraction buffer (0.5 % Triton X-100, p H 7. 4, 4°C) Combine 10 mL of I O X stock buffer, 0.5 mL of P M S F, 3 mL of E D T A and 5 mL of freshly prepared 10% Triton X-100. I mL of PMSF, 3 mL of EDTA and 5 mL of freshly prepared 10% Triton X-100 were added to 100 mL. (6) Nucleus protein extraction buffer (1 % Tween-40/0.5 % 11-deoxycorticosterone (DOC), p H7.4,4°C): dissolve 0-5 g of DOC and I mL of Tween-40 in 2.5 mL of IOX stock buffer (heat to dissolve if necessary), combine and add 5 mL of 10 X stock buffer and IOX stock buffer. 10 X stock buffer and I m L P M S F , and add water to 100 m L. (7) Cellular Skeleton Dissolution Buffer (5% SDS, 10 mmol/L sodium phosphate, pH 7.4): For a non-reducing buffer, dissolve ∼0.5 g of SDS in 5 mL of 20 m m o l / L sodium phosphate buffer (p H 7.4). Add water to a volume of 10 m L. For denaturing buffer, add I m L of mercaptoethanol. Finally, add water to make appropriate adjustments. Steps (1) Rinse the cells with ice-bath-cooled cytosolic protein extraction buffer (5 vol/g wet weight, resuspend the cells by slight vortex shaking) or monolayer the cells (I m L/T 2 5 fine-necked vials). (2) Incubate the cells on ice and gently shake (platform mixer) until 9 5 to 10 % of the cells have been shown to be permeable (5 to 10 m i n ) by trypan blue staining assay. (3) For suspension cultures, centrifuge the extracted mixture at 500 g and remove the supernatant. (4) For monolayers, tilt the flask and pipette out the extracts (cytosolic protein-rich fractions) and store at 70°C. (5) Carefully resuspend the undissolved particles using the same volume of ice-bath cooled Cell Membrane Protein Extraction Buffer as used for the cytoplasmic protein extract (5 times the volume of the wet weight of the initial material) to obtain a homogeneous suspension. (6) For monolayers of cells, add I m L of Cell Membrane Protein Extraction Buffer to each T25 vial (approximately 5 X IO6 cells). (7) Incubate on ice with gentle shaking for 30 m i n . (8) Centrifuge the suspension at 5000 g for 10 m i n to remove extracts (fractions rich in cell membrane and organelle proteins) or decant the monolayer. (9) Dispense in aliquots and store at 70°C. (10) Resuspend the insoluble pellet in suspension culture with half of the nuclear protein extraction buffer used for cell membrane protein extraction. (11) Centrifuge at 4°C and 6000 g for 10 m i n to allow the residual insoluble fraction of the detergent to form a precipitate and remove the extract (the extract rich in cytosolic proteins). (12) For each monolayer of cells equivalent to the volume of T 2 5 fine-necked vials, extracts were made using 0.5 ~ I m L of cytosolic protein extraction buffer. (13) Aliquots are dispensed and stored at 70°C. (14) For detergent-insoluble particles, rinse with ice-bath-cooled PBS (p H 7.4, I.2 m m o l / L PMSF) and mechanically break down the DNA by resuspension (PTFE resin/glass homogenizer) and set up a 12,000 g centrifugation for 20 m in at 4°C or by adding 1000 units of Benzonase nuclease (EMD/Novagen) for enzymatic digestion of the nucleic acids. Nucleic acids were enzymatically digested by adding 1000 units of Benzonase (EMD/Novagen). (15) The precipitate from the suspension culture is washed once with 90% acetone at 20°C, lyophilized, and weighed in a tared centrifuge tube. The resulting samples are stored at 70°C. (16) For monolayers of cells, an in situ (in w i w ) wash is performed with P B S. The residual descaler-insoluble fraction (cytoskeleton) may be resuspended by adding directly to 0.5-1 ml of β-mercaptoethanol-free nondenaturing cytoskeletal lysis buffer. Store at -70°C. Materials and Equipment (1) TNE buffer [25 mmol/L Tris-HCl (pH 7.5), 50 m mol/L NaCl, 5 mmol/L EDTA] containing 1 % (Translator's note: it seems that the original text contains l% Triton X-100 in error). (2) Protease and phosphatase inhibitors (calbiochem). (3) Nycodenz. (4) Centrifuges of the outward opening type and the corresponding rotors. (5) Heterogeneous homocrystalline polymer centrifuge tubes. Steps (1) Spread 2. OXIO7 ~ 3. OXIO7 cultured cells (adherent or suspended) in a cell culture flask. (2) Wash the cells 2 times with pre-treated P B S . (3) Cells were treated with β-MCD (lO m m ol/L) for 45 m in and/or cholesterol for I h at 37°C in serum-free medium. (4) Stimulated cells were infected after treatment or at 72 h, if needed. (5) Cells were lysed using a pre-cooled TNE buffer containing 1 % T r i t o n X -I O O 7 50 bucket with protease and phosphatase inhibitors and incubated for 30 m i n in an ice bath. (6) Adopt 70% Nycodone z 1500 juL to mix with the lysate, then dissolve in TNE buffer. (7) The mixture was added to the bottom of a 4 mL ultracentrifuge tube of heterogeneous homocrystalline polymer. (8) Add TNE buffer to the bottom of the centrifuge tube to prepare 25%, 21.5%, 18%, 15% and 8% Ny-codenz and mix well. (9) Centrifuge at 4°C for 4 h at 200,000 g using an open rotor. (10) A total of 350 buckets from the top to the bottom of each centrifuge tube are analyzed by WESTERN BLOOT analysis. (11) Analyze the W e s t e r n B l o t results to verify the presence of foveal protein 1 positivity (3 to 6 fractions) or the presence of detergent-resistant cell membrane fractions. Preparation of washed cell nucleus pellets The isolation of nucleosomes and free histones requires cleaned cell nuclei as initial raw material. In this scheme, nuclei are obtained by treating cultured cells with homogenization and the non-ionizing decontaminant N P -40. The cells were washed several times using a buffer containing the decontaminant to remove the cell membranes to obtain particles containing relatively clean nuclei. Subsequently, NACL extraction is used to remove most of the proteins that are slightly bound to the chromatin. However, washing with NP-40 can result in a loss of extract activity relative to other methods of nucleus extraction. If both nuclei and chromatin are to be extracted, then the extract should be Materials and equipment (1) 2 m o l / L N a C l (2) Nucleus Precipitation Buffer 2 (NPB): 0.6 m o l / L K C l , 10 % (V / V) glycerol. (3) D0u n c e homogenizer and B-stick. (4) Nucleus Precipitation Buffer (NPB). (5) Store at 4°C for several weeks. Before use, add 3 m m o l / L 2-mercaptoethanol, 0.5 m m m o l / L PMSF, I um m m o l / L pepstatin A, and 1 p m m o l / L Ieupeptin. (6) Ieupeptin. An aqueous solution of I m m o l /L is prepared as a storage solution and can be stored at 20°C for 1 year. (7) Lysis buffer (L B ). 20 m m o l / L H E P E S , p H 7. 5; 0-25 m m o l / L L Leptospermum; 3 m m m o l / L M g C l 2 ; 0.5 % (V /V ) Nonidet P -40 (N P -40). (8) Store at 4°C for several weeks. Before use, add: 3 mMol/L 2-mercaptoethanol, 0.5 mMol/L PMSF, I umol/L pepsin A, 1 fxmol/L leupeptin peptide. Note: All solutions were placed in an ice bath or at 4°C unless otherwise indicated. Steps (1) 3 X I O9 cells (H e L a ) are washed twice with I L P B S . (2) The granular precipitate was resuspended using 40 m L of L y sis buffer. (3) Lysed the cells with a D o u n c e homogenizer with a B bar set to approximately 1 5 shocks. Monitor cell lysis by light microscopy. (4) Centrifuge at 4°C and 3000 ming for 15 m i n . (5) Resuspend the pellet twice with Lysis buffer and once with NPB. (6) Resuspend the nuclei with 2 times the volume of NPB and measure the total volume of the suspension. (7) While stirring gently, add 1 total volume of NPB2 dropwise. (8) Stir gently at 4°C for 10 m i n . (9) Centrifuge for 30 m i n at 4°C and 1750 volume to obtain the nucleus pellet precipitate. (10) Freeze the pellet with liquid nitrogen or dry ice and store at 1 80°C for more than 1 year. Materials and Equipment (1) Nucleus pellet precipitation (as described above). (2) Hydroxyapatite ( H P ) buffer with and without 2. 5 mol/L N a C l (as described below). (3) BioGel H T P powder (Bio-Rad) with an adsorption capacity of 0. 6 m g of D N A per gram of dry powder. (4) 2 c m X 15 c m chromatography column and accessories. (5) Centriprep-IO concentrator (Amicon; optional). (6) Other reagents and equipment for the detection of protein concentration and SD & PAGE. (7) H P buffer: 50 m m o l /L sodium phosphate ( p H 6. 8), 0. 6 m m o l / L N a C L can be stored at 4°C for several weeks. Add 1 m m o l / L 2-mercaptoethanol, 0.5 m m o l / L PMSF before use. Procedure (1) Resuspend approximately 2 m L of nuclei pellet containing approximately 6 m g D N A using 25 m L of H P buffer. (2) Warmly stir at 4°C for 10 m i n . To avoid protein hydrolysis, add I u mol/L pepsin and I um o l/L leucine peptide to the HP buffer. (3) Add 10 g of BioGel HTP dry powder (hydroxyapatite). (4) Prepare an I : 1 slurry by adding H P buffer. Pour into a 2 c m X 15 c m chromatography column and collect the eluate. (5) Wash the resin with 300 m L of H P buffer (30 m L / h ). The eluate obtained in step (3) and the first column volume wash contains H l . (6) Elute the core histones with HP buffer containing 2.5 mOl/L NACL and collect 8 mL of the fraction. (7) SDS--PAGE analysis of core histone purity. (8) Concentrate core histones to 10 m g /m L using a Centriprep-l O concentrator if necessary. (9) Dispense into aliquots, freeze on dry ice, and store at 1 80°C for up to 4 years. Materials and Equipment (1) Nucleus Buffer Liquid I (N B l ) : 25 m m o l / L H E P E S ( p H 7. 9), 5 m m o l / L K C 1, 0.5 m m o l / L M g C l 2, I m m o l / L D T T , 100 m m o l / L P M S F , volume set to 100 m L (2) Nucleus buffer 2 (N B 2): 100 m L N B 1 + 1 m L N P -40 [ 1 % polyethylene glycol phenyl ether (I G E -P A L )]. (3) Nucleus Buffer 3 (N B 3): 1:1 mixture of N B 1 and N B 2. (4) Nucleus buffer 4 (N B 4): 25 m m o l / L H E P E S ( p H 7. 9) ,350 m m o l / L N a Cl (5) Brilliant protease inhibiting peptide, PMSF, peptidase, gastric enzyme inhibitor and D T (Calbiochem). (6) IGEPAL CA-630 (Sigma). Note: All components (buffers, protease inhibitors, etc.) must be kept in an ice bath during the procedure. Steps The following protocol is suitable for the manipulation of IO7 cells. (1) Remove the medium and add I m L of ice-bath cooled PBS. (2) Scrape the cells into the PBS and transfer them to an eppendorf tube and centrifuge at 10,000 g for 5 mIn. (3) Remove the supernatant and resuspend the precipitated pellet with 200 of N B l (can be scaled up or down). (4) Add 2 000 drams of NB2 and spin at 4°C for 15 m i n . (5) Centrifuge at 2500 g for a minimum of 10 minutes. (6) Transfer the supernatant to a new eppendorf tube (enriched with cytoplasmic protein fraction). (7) Add 100 ) u L of N B 3 again and mix gently. Transfer the wash solution to a new eppendorf tube (enriched with cytoplasmic protein fraction). (8) Add 500 p L of N B 4 , and spin at 4°C for I h . (9) Spin for 10 m i n at maximum rate and the resulting supernatant is the nucleus protein sample. Materials and Equipment (1) protein A/protein G coupled to stationary phase in a buffer of H E P E S containing 1 % B S A , 100 m m o l / L N a C l and 0. 0 1 % N a N 3 , p H 7. 0 . (2) Monoclonal or polyclonal antibodies. (3) Wash buffer worker (w a s h buffer worker, W B 1) :20 m m o l / L H E P E S , p H 7. B S A . (4) Paint wash buffer I K w a s h buffer II, " W B 2) :20 m m o l / L H E P E S , p H 7.0; 100 m m o l / L N a C l . (5) Elution buffer (E B ): 0.1% S D S , 50 m m o l / L Tris, p H 6. 5; 100 _ ol/L D T T and 10 % glycerol. Steps Pre-clarify the cell lysate as described above. (1) Incubate the lysate, antibody (based on information provided by the supplier), protein A /prot e i n G and stationary phase (30 M L of 1 : 1 slurry) at 4 °C with gentle stirring overnight. (2) Centrifuge at 4°C, 500 g■ for 5 min. (3) Wash the pellet with 500 of Wash Buffer I and repeat 3 times. (4) Wash the pellet precipitate with Wash Buffer II at 500, repeat 2 times. (5) Elute the encapsulated vesicles/organelles with 30uL of Elution Buffer, under mild extraction conditions if required. (6) Centrifuge I m i n at 5000 g - . The resulting supernatant is a solution enriched with lattice protein-coated vesicles (Fig. 19.6). For more product details, please visit Aladdin Scientific website.
(1) Sucrose solution: concentrations 1 5 %, 20 %, 25 %, 27.5 %, 30 % and 35 % (m/V).
The lysate was pre-clarified by centrifugation at 500 g at 4°C for 5 m i n .
Combine with the remaining 6 mL of IO X reserve buffer and 5 mL of EDTA. Add water to volume to 100 m L.
Nuclei should be extracted as described below, and the resulting nucleus pellet can be used to prepare chromatin (Schnitzler, 2001).
20 m m o l / L H E P E S , p H 7. 5; 3 m m o l / L M g C l 2 ; ○.2 r n m o l / L E G T A
(6) to the cytoplasmic protein solution prepared in step (6). If the pellet precipitate is disturbed, a short centrifugation may be necessary.
