B-PER™ with Enzymes Bacterial Protein Extraction Kit, 250 mL - FAQs

在富营养培养基（高糖）中生长的细菌往往难以裂解。您可以尝试减少葡萄糖浓度。B-PER试剂能够裂解包括E. coli，Acetinobacter，Archaebacteria，S. aureus，H. pylori，昆虫杆状病毒，线虫和昆虫细胞在内的细菌和细胞。B-PER试剂可以裂解一些革兰氏阳性菌，但是裂解革兰氏阴性细菌的效率通常更高。如果对某一特定细菌菌株裂解效果不佳，则请在裂解前冷冻细菌。添加溶菌酶是最有效的提取手段，但是对于一些过表达蛋白则不需要加入溶菌酶。

B-PER II Reagent contains more detergent making it ideal for extracting proteins from small bacterial cultures with less than 20 mL in volume.

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B-PER Reagent utilizes a proprietary, mild nonionic detergent in a 20 mM Tris HCl, pH 7.5 buffer. No enzymatic components are present in the B-PER solution itself (B-PER Complete Bacterial Protein Extraction Reagent, Cat. No. 89821, 89822 does contain enzymes). Depending on your particular protein, you may need to add components such as salt, lysozyme, protease inhibitors, reducing agents and chelating agents. Other ready-to-use formats include B-PER II Bacterial Protein Extraction Reagent (2X), B-PER Reagent (in Phosphate Buffer), and B-PER with Enzymes Bacterial Protein Extraction Kit.

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For 1 gram of cells add 4 mL of B-PER Reagent.

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While this is protein-dependent, many proteins have been successfully tested in downstream applications (reporter assays, immunoprecipitation and beta-Gal assays), including GST and 6xHis proteins. Otherwise, samples can be diluted or dialyzed to remove any interfering substances.

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This is often an indication of the presence of large amounts of DNA in the extract. This viscosity will be greatly decreased upon addition of DNase I (Cat. No. 90083).

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It is found in the pellet after the first round of extraction of soluble proteins, so it should not be present in the final protein preparation.

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You can add up to a final concentration of 0.5 M salt.

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Inclusion bodies are easily purified using B-PER Reagent. To recover your protein from inclusion bodies, however, we recommend the use of Thermo Scientific Inclusion Body Solubilization Reagent (Cat. No. 78115).

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SDS-PAGE examination of the protein pellet, as well as the lysate, will reveal whether the protein of interest is present in inclusion bodies or if it is soluble.

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Using 1 L of E.coli culture with an OD600 of approximately 2 will give approximately 8 grams wet cells. For 8 g cells, use 40-50 mL of B-PER Reagent.

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The Thermo Scientific BCA Protein Assay Kit (Cat. No. 23225, 23227) works very well to detect proteins extracted with B-PER Reagent. The Coomassie Plus Protein Assay (Cat. No. 23236) can be used with B-PER Reagent Protein extractions, however when using B-PER with PBS or B-PER II reagents, dilute the protein preparation two-to-four fold before assaying with Coomassie Plus or Prepare sample with Compat-Able Protein Assay Preparation Reagent Set (Cat. No. 23215). Finally, Thermo Scientific 660 nm Assay Kit (Cat. No. 22662) can also be used if protein extract is diluted two fold.

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B-PER Reagent is designed to replace sonication, which cannot fully recover soluble proteins or regain inclusion bodies. Although freezing the bacterial pellets is not usually necessary, it appears to increase protein yield in some bacteria that are traditionally difficult to lyse.

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B-PER Reagent is designed to be used with, among other applications, 6xHis and GST fusion protein purification. When using B-PER Reagent with other IMAC or glutathione columns, use a dilution of at least one-to-one B-PER Reagent in the binding buffer recommended for that column. This will decrease the possibility of incompatibility of buffer components.

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The B-PER Bacterial Protein Extraction Reagent is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended (ambient temperature). Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

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The B-PER Reagent solution contains a proprietary, mild, non-ionic detergent in 20 mM Tris-HCl, pH 7.5. It effectively disrupts cells and solubilizes native or recombinant proteins without denaturation. The reagent creates holes in the cell membrane that will leak out cytosolic proteins. The sample may become very viscous when the bacterial chromosome is released. We recommend adding DNAse I (Cat. No. 90083) to the reagent to reduce viscosity. For better lysis efficiency and if there are inclusion bodies, we recommend adding Lysozyme (Cat. No. 90082) to the reagent. Alternatively, you may purchase the B-PER Bacterial Protein Extraction Reagent with Enzymes Kit (Cat. No. 90078 or 90079) that includes the B-PER Bacterial Protein Extraction Reagent, DNase I, and Lysozyme.

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Bacteria grown in rich medium (high glucose) are difficult to lyse. Try reducing the glucose concentration. Bacteria and organisms lysed with B-PER Reagent include E. coli, Acetinobacter, Archaebacteria, S. aureus, H. pylori, Baculovirus, nematodes, and insect cells. B-PER Reagent can lyse some gram-positive bacteria, but tends to be more efficient with gram-negative bacteria. If lysis is inefficient for a particular bacterial strain, freeze cells before extraction. Add lysozyme for the most efficient extraction, however, for some over-expressed proteins, the addition of lysozyme is not required.

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Many proteins spontaneously refold into their native, functional structures when the denaturing solubilization reagents are removed by dialysis. Other proteins, however, will fold into non-functional and even insoluble forms by this process. In such cases, specialized sets of buffer conditions must be tested to identify those that promote the highest possible yield of properly refolded protein. See our Pierce Protein Refolding Kit (Cat. No. 89867).

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Add DNase I to the B-PER solution. If DNase was added, the enzyme may have low activity or be inactive. You can add Mg2+ ions to 2 mM final concentration, add more DNase I to the reagent, or purchase new enzyme. Alternatively, use B- PER Complete reagent, an all-in-one formulation that combines a lysis reagent with lysozyme and a universal nuclease to enable mild extraction from both gram-negative and gram-positive bacteria.

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The temperature may be too low; warm the culture and other reagents to room temperature. The lysozyme may have low activity or be inactive. Add more lysozyme to the reagent or purchase new enzyme.

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We recommend using B-PER Complete Bacterial Protein Extraction Reagent, an all-in-one formulation that combines a lysis reagent with lysozyme and a universal nuclease to enable mild extraction from both gram-negative and gram- positive bacteria. If the addition of lysozyme and DNase I might interfere with your downstream application, we recommend using other B-PER reagents. See details of the various B-PER reagents we offer.

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Some overexpressed proteins can be insoluble, misfolded, or expressed in inclusion bodies. The best way to obtain soluble protein is to adjust the expression conditions; however, if this is not possible, the Inclusion Body Solubilization Reagent (Cat. No. 78115) can be used.

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See details of the various B-PER reagents we offer (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction/b-per-bacterial-cell-lysis-reagents.html).

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We offer the B-PER Bacterial Protein Extraction Reagent for lysis of bacterial cells.

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