Protein Extraction Kits

• Total Protein Extraction Kit
• Compartmental Protein Extraction Kit

Total Protein Extraction Kit

The Total Protein Extraction Kit is an excellent tool for the initial purification and preparation of proteins from any tissues or cells. Total protein isolated by this kit is native and can be used for downstream applications including SDS-PAGE, Western blotting, gel mobility shift assays, protein assays and other procedures. The kit is easy to use: no scraping, no freeze-thaw cycles, and no sonication.

Features

• Isolate protein from large samples
• Rapid isolation without freeze/thaw cycles or sonication
• Isolate protein either from tissues or from cultured cells
• Isolated protein is intact (some proteins purified by this kit could be over 200 kDa in mass)

Applications

50 µg of total proteins on 4-20% gradient SDS-PAGE gel. Total proteins were isolated from 8 different human tissues by the Total protein extraction kit. Lane 1: Liver; Lane 2: Kidney; Lane 3: Brain; Lane 4: Skeletal Muscle; Lane 5: Spleen; Lane 6: Lung; Lane 7: Placenta; Lane 8: Stomache.

Description

Kit Components

Protocol

1. Dilute 50X PI solution to 1X PI in TM buffer keeping the solution on ice.
2. Weigh certain amount of tissues and chop them into small pieces.
3. Keep the tissues on dry ice.
4. Add 1X PI in TM buffer to the tissue at 2.5 mL per gram of tissue or per 25 million cells, and put in ice for 5 minutes.
5. Homogenize the tissue or cells for 20 seconds and then put on dry ice for 15 seconds.
6. Homogenize the tissue or cells for the second 20 seconds. (A third 20 seconds homogenization may be required if the tissue or cells are not well homogenized.)
7. Rotate the homogenized tissue or cells at 4°C for 20 minutes.
8. Centrifuge at 11,000 rpm at 4°C for 20 minutes.
9. Collect the supernatant.
10. Determine the concentration of the total proteins.

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Compartmental Protein Extraction Kit

For extracting Cytoplasmic, Nuclear, Membrane, and Cytoskeletal proteins from tissues and cells.

One of the challenges of functional proteomics is separation of complex protein mixtures for quantitative and differential subcellular localization analysis. This necessitates standardized and repeatable procedures to isolate subcellular proteomes from tissues and cells. Millipore’s Compartmental Protein Extraction Kit addresses the challenge by providing an innovative, easy-to-perform, and cost-effective method to sequentially isolate cytoplasmic, nuclear, membrane, and cytoskeletal proteins from mammalian tissues and cells, based on a proprietary technique.

Features

• Convenient – Provides a complete set of components for stepwise preparation of cytoplasmic, nuclear, membrane, and cytoskeleton proteins from mammalian tissues and cultured cells
• Fast – Isolate four compartmental proteomes in less than three hours
• Reliable – High quality reproducible separation of subcellular protein fractions as verified by clearly distinct protein patterns and precise subcellular localizations of marker proteins
• Pure – Minimal cross contaminations
• Simple – Easy to use compared to other methods, such as differential centrifugation

Description

Kit Components

• Buffer C: 1 x 18 mL of HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium OrthoVanadate (store at 2–8°C).
• Buffer W: 1 x 50 mL of HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium OrthoVanadate (store at 2–8°C).
• Buffer N: 6 mL of HEPES (pH 7.9), MgCl2, NaCl, EDTA, Glycerol, Sodium OrthoVanadate (store at 2–8°C).
• Buffer M: 6 mL of HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate, NP-40, Sodium OrthoVanadate (store at 2–8°C).
• Buffer CS: 3 mL of Pipes (pH 6.8), MgCl2, NaCl, EDTA, Sucrose, SDS, Sodium Ortho-Vanadate (store at 2–8°C).
• 50x PI: 0.66 mL cocktail of protease inhibitors (store at –20°C).

Protocol

For adherent cells, we recommend adding Buffer C (the extraction buffer) directly to the plates, and scrapping the cells loose with a plastic cell scraper. The mixture is then pipetted into tubes and vortexed or rocked, cooling on ice frequently.

Note: Cultures should be rinsed well with PBS prior to the addition of the extraction buffer. Failure will result in high levels of FBS and protein contamination from the cell culture media, which can make Western blot analysis of the lysates difficult in some casesFor suspension cells, we recommend adding the extraction buffer directly to the washed, prepared cell pellet, then gently pipeting or vortexing the solution. Store the cell lysate on ice and use precooled solutions for best results.

Note: For buffers C, N, M and CS, add 50X PI to working concentration of 1X, before use. Prewarm the CS buffer to room temperature to ensure that SDS is completely in solution.

Total protein and compartmental proteins extracted from rat colon tissue A using Millipore Total Protein Extraction kit and the Compartmental Protein Extractin kit were submitted to SDS-PAGE in 5 identical gels. (A) Coomassie staining of one piece of gel indicated distinct protein pattern of respective fractions. (B) Immunoblotting of PVDF membranes transferred from four other pieces of gel against cytoplasmic marker protein GAPDH, nuclear marker protein Histone H1, membrane marker protein EGFR, and cytoskeletal marker vimentin assigned the majorities of the marker proteins to their expected compartmental fractions. Lane 1: Total protein; Lane 2: Cytoplasmic Protein; Lane 3: Nuclear Protein; Lane 4: Membrane Protein; Lane 5: Cytoskeletal Protein.

1. Weigh a small amount of desired tissue (0–1 gm), and then cut tissue block into small pieces on a prechilled plate, then pipette ice cold Buffer C using 2.0 mL per gram tissue or per 20 million cells. More buffer can be used if necessary. Lowering the amount used is not recommended because if the solution becomes too thick this will compromise the fractionation of the cellular components. Homogenize* the tissue or cells at moderate speed (e.g. speed 4) by rotation or vortexing, for 20 seconds. Let it stand on ice for a few seconds, repeat homogenization twice more, or until solution is uniformly homogenized.
2. Rotate, or gently mix the mixture at 4°C for 20 minutes.
3. Centrifuge sample at 18,000 g at 4°C for 20 minutes. Remove and save the supernatant into another tube. The cytoplasmic proteins are in the supernatant. The pellet contains membranes, nuclei, and cytoskeletal fractions. Keep storage tubes on ice.
4. Wash and resuspend the pellet with 4 mL of ice cold buffer W per gram of tissue/20 million cells, rotate or mix at 4°C for 5 minutes. Centrifuge again at 18,000 g at 4°C for 20 minutes. Carefully pour off the supernatant and discard, wipe the edge of the tube with a tissue if necessary. This wash step simply helps to reduce cross-contamination with cytoplasmic proteins and it can be repeated for increased fidelity if desired.
5. Add 1.0 mL of ice cold buffer N per gram of tissue/20 million cells to the pellet and resuspend the pellet from step 4, rotate or mix at 4°C for 20 minutes. Spin at 18,000 g at 4°C for 20 minutes. Remove and save the supernatant in to another tube. The nuclear proteins** are in this supernatant. Keep storage tubes on ice.
6. Add 1.0 mL of ice cold buffer M per gram of tissue/20 million cells to the pellet and resuspend, rotate or mix at 4°C for 20 minutes. Spin at 18,000 g at 4°C for 20 minutes. Remove and save the supernatant into another tube. The membrane proteins are in this supernatant. Place tube with pellet on ice.
7. Pre-warm an aliquot of CS buffer to room temperature or 37°C to make it clear, and add 0.5 mL of the room temperature CS buffer per gram of tissue/20 million cells to the pellet from step 6 and resuspend the pellet. Rotate or mix the solution at room temperature for 20 minutes. Spin at 18,000 g at 4°C for 20 minutes. Remove and save the supernatant in to another tube on ice. Place tube with pellet on ice.
8. Immediately wash and resuspend the pellet from step 7 with 1.5 mL of ice cold buffer C per gram of tissue/20 million cells, rotate or mix at 4°C for 5 minutes. Spin at 18,000 g at 4°C for 20 minutes, save the supernatant on ice.
9. Combine the supernatants from step 7 and step 8. The cytoskeletal proteins are in this mixed solution.
10. Measure the protein concentrations of the four fractions with a detergent compatible protein concentration kit. Aliquot and label the proteins properly and store at –70°C. For best results avoid repeated freeze-thaws of the protein extracts.

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