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AlleleExtract Protein Extraction Reagent, an effective and gentle method

for extracting proteins from cells.


he AlleleExtract Protein Extraction Reagent product line offers simple and effective methods for isolation of proteins from bacterial and mammalian cells. The AlleleExtract-M is for extraction of proteins from mammalian cells, while AlleleExtract-B is suitable for bacterial cells. lleleExtract-M Mammalian Protein Extraction Reagent provides an effective and gentle method for extracting proteins from mammalian cells. This reagent uses a non-ionic detergent in either PBS (products with catalogue number ending with P) or 25 mM Tris, pH 7.4, and 250 mM (products with catalogue number ending with T) to extract nuclear and cytoplasmic protein from cultured cell lines. AlleleExtract-M may be used conjunctively with reporter assays, immunoassays, and protein purification. It is easily digested with protein assays, such as Western blotting, Coomassie Blue, Bradford, and BCA Protein Assays. lleleExtract-B Protein Reagent provides a single-step method of gently extracting proteins from a wide range of bacterial cells. Using this reagent eliminates the need for repeated sonication and freezing/ thawing steps that are commonly used to release recombinant proteins from E coli. Proteins so extracted are suitable for many affinity purification and analysis methods, e.g. GST and 6xHis purification systems. If preferred, DNAse I, lysozyme, protease inhibitor, salts, etc. may be added to the reagent. AlleleExtract-B Protein Reagent offers several fold increase in the yield of soluble proteins when compared with other commercial lysis systems.
or Research Use Only. Not for Diagnostic or Therapeutic Use. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele Biotech is strictly prohibited

Features

Considerations
For AlleleExtract-B Reagent
1.) E. coli strain: The AlleleExtract-B Protein Extraction Reagent is effective for extracting from BL21 E. coli strains as well as most commonly used lab strains. T7 lysozyme activity enhances extraction in the commonly used pLysS or pLysE strains. Note: Lysates from these strains have higher viscosity, DNase I may be needed to refine lysates. 2.) Buffer compatibility: For most proteins produced in bacteria, phosphate buffers are recommended for purification applications following protein extraction with the AlleleExtract-B Protein Reagent. For certain proteins, however, improved extraction and purification can be accomplished with AlleleExtract-B Protein Reagent in 20 mM Tris HCl (pH 7.5). 3.) Use of lysozyme: Although AlleleExtract-B Protein Reagent may be used conjunctively with lysozyme, lysozyme is not necessary to extract soluble proteins. However, when isolating inclusion bodies, lysozyme should be used to digest cell debris to release inclusion bodies.

ll Extract-M
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method for protein extraction from mammalian cells; eliminates cell scraping from culture dish.

Highly effective and gentle

Fully dialyzable and may be used Extract proteins from mammalian

conjunctively with subsequent assay systems.

cells for most molecular biology applications including Western Blotting, Coomassie Blue, Bradford, BCA Protein Assays, and others. lleleExtract-B

Nonionic detergent in either PBS or Tris based buffer. Eliminates mechanical disruption and provides a gentle and efficient method for extracting proteins while achieving high yields of soluble proteins. May be used for soluble and insoluble protein extraction from bacterial cell lysates
systems

Compatible with most purification

Box 1 | Product List


AlleleExtract-M Protein Extraction Reagent
ABP-PA-AXMR125P ABP-PA-AXMR500P ABP-PA-AXMR125T ABP-PA-AXMR500T
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125 mL in PBS 500 mL in PBS 125 mL in Tris 500 mL in Tris

All Extract-B Protein Extraction Reagent


ABP-PA-AXBR125P ABP-PA-AXBR500P ABP-PA-AXBR125T ABP-PA-AXBR500T 125 mL in PBS 500 mL in PBS 125 mL in Tris 500 mL in Tris

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Protocols
AlleleExtract-B Reagent For Obtaining Proteins in Soluble Fraction: 1. 2. 3. Collect cells from 40-250 mL bacterial culture (OD600 1.5-3.0) at 3,500 x g for 10 minutes. For small cultures of 1.5-3 ml, collect at 10,000 rpm in a microcentrifuge. Decant or aspirate all media. Although cells can be used fresh or frozen at -80 C, freezing cells often aids the extraction. Resuspend pellet from 250 ml of culture in 15-20 ml of AlleleExtract-B Protein Reagent (for 40 ml culture, use 3 ml; 1.5 ml culture, use 150 L of AlleleExtract-B Reagent) by carefully pipetting up and down until suspension is homogenous. Rotate tube for additional 10 minutes; do not vortex. If Bl21(DE3) pLysS strain is used, add 1 kU/ml of DNase I. Centrifuge at 27,000 x g for 15 minutes. (or 14,000 rpm in microcentrifuge for 5 minutes with pellets from small cultures) to separate soluble and insoluble portions. The supernatant is the soluble protein. Transfer supernatant to a fresh tube and resuspend insoluble fraction in 2 volumes of AlleleExtract-B Reagent used in Step 3. *Use 10 L each of the soluble and insoluble fraction for SDSPAGE and/or Western blotting to determine protein solubility. 2. Add suitable amount of C to sample plate.* *Note: Protease inhibitors may be added to AlleleExtract-M if desired.

Box 2 |Suggested volume of AlleleExtract-M Reagent for culture plates


Plate Type Volume of AlleleExtract-M Reagent
100 mm 50 mm 6-well plate 24-well plate 96-well plate 500 - 1000 L per well 250 - 500 L per well 200 - 400 L per well 100 - 200 L per well 50 - 100 L per well

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Incubate for 10 minutes at 25 C with mild agitation or rotation. Note: Some cell types transfected with large amounts of DNA and transfection reagent may need a single round of freezing for 10 minutes at -80 C recommended. Gather lysate and transport to a microcentrifuge tube. Centrifuge samples at 14,000 x g for 5 minutes to pellet cell remains. Note: Steps 4-5 are optional. Lysate may be used for assays with cell remains unremoved. Transfer lysate to clean tube for further analysis.

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Recommended Suggestions for Lysis of Suspension Mammalian Cells: 6. 7. Follow steps 1 through 4 for soluble protein extraction. Resuspend pellet from step 4 in 8M urea, in either PBS or Tris buffer (20mM, pH7.5). The same amount of volume of AlleleExtract-B Reagent used in step 3 above. Vortex for 1 minute and incubate at room temperature for 5 minutes. The pellet is normally resolved by this step. Proceed to purifying protein as normal. The presence of urea does not affect most commonly used dialyzing against 4M, 2M, 0M urea in either PBS or Tris for about 4hours to overnight at each step. Beware that some proteins will become insoluble again as urea is being removed. 1. 2. Pellet the cell suspension by centrifugation at 3,000 x g for 10 minutes. Carefully remove supernatant and discard. Optional Wash: Wash cells once with PBS if culture medium contains phenol red or other reagents that may interfere with protein analysis. Centrifuge washed cells at 3,000 x g for 10 minutes. Discard supernatant Add AlleleExtract-M Reagent to cell pellet. Use 1 mL of AlleleExtract-M Reagent for each 100 mg (from approximately100 ml culture) of wet cell pellet. If
ele Extract-M Reagent and resuspend cell pellet by pipetting. Add remainder of AlleleExtract-M Reagent to cell suspension. Shake cell suspension mildly for 10 minutes. Remove cell remains by centrifugation at 3,000 x g for 15 minutes. Transfer supernatant to clean tube for further analysis.

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AlleleExtract-M Reagent Recommended Suggestions for Lysis of Adherent Mammalian Cells: 1. Gently remove the culture medium from adherent cells. wash cells once with PBS if culture medium contains phenol red or other reagents that may interfere with protein analysis.

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