Thermo Scientific™ B-PER™ Bacterial Protein Extraction Reagent

Product Code.	Quantity	unitSize
10541104	500 mL	500mL
11834091	165 mL	165mL
10733485	250 mL	250mL

Product Code. 10541104 Supplier Thermo Scientific™ Supplier No. 78248

Description

Efficiently lyse E.coli and other bacterial cells and gently extract native proteins with these nonionic detergent solutions.

Perform efficient, gentle lysis and extraction of E.coli and other bacterial cells with Thermo Scientific™ B-PER Bacterial Protein Extraction Reagents.

These easy-to-use cell lysis reagents are nonionic detergent solutions that effectively disrupt cells and solubilize native or recombinant proteins without denaturation. Certain formulations include DNase I and lysozyme enzyme supplements to improve yield of large molecular weight proteins that are difficult to purify. B-PER Reagents are compatible with the addition of standard protease inhibitors and with downstream protein purification methods, such as GST purification and His-tagged immobilized metal affinity chromatography (IMAC).

Highlights:

• Ready to use – B-PER Reagents provide one-step E.coli cell lysis by a mild, nonionic detergent (proprietary) in your choice of Tris or phosphate buffer formulations
• Fast and simple – just add B-PER Reagent to a bacterial pellet, shake for 10 minutes and recover soluble proteins after pelleting the cell debris
• Excellent yields – recover both soluble and insoluble recombinant protein from bacterial lysates and purify inclusion bodies to near-homogeneous levels
• Flexible – B-PER Reagents are suitable for any scale of protein extraction and are available in phosphate and 1X and 2X Tris formulations, with and without enzymes

• Compatible – completely compatible with addition of protease inhibitor cocktails, and resulting protein extract can be used in protein assays, typical affinity purification methods (e.g., GST, 6xHis) and other applications

Convenient, ready-to-use formats:

• B-PER and B-PER II (2X) Reagents are free of enzymatic components
• B-PER Reagent (in phosphate buffer) is an amine-free formulation for direct compatibility with labeling and crosslinking
• B-PER with Enzymes and B-PER Direct with Enzymes include lysozyme and DNase I for improved lysis and recovery

Includes:

Reagent packages are available in Tris and phosphate buffer formulations; Kits contain Tris-based B-PER Reagent plus lysozyme and DNase I enzymes.

Specifications

• Description: B-PER Bacterial Protein Extraction Reagent
• Quantity: 500 mL
• Product Type: Bacterial Protein Extraction Reagent
• Volume (Metric): 500 mL
• Content And Storage: Store at room temperature.
• Product Line: B-PER

Frequently Asked Questions (FAQs)

What is the difference between B-PER and B-PER II Reagent?

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

What is the composition of B-PER Reagent?

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.

What is the volume of B-PER Extraction Reagent to use per gram weight of wet cells?

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

Will my protein be in its native conformation after B-PER Reagent extraction?

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.

Why is my protein extract so viscous after B-PER Reagent extraction?

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).

Where is the DNA found when extracting proteins with B-PER Reagent?

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.

What is the maximum amount of NaCl that I can add to B-PER Reagent for purification of my salt-dependent protein?

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

My protein is insoluble. Can I purify it using B-PER Reagent?

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).

How will I know if my recombinant protein is soluble or insoluble after extraction with B-PER Reagent?

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.

How much B-PER Reagent should I use for large-scale preps?

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.

How can I measure the amount of protein I have extracted with B-PER reagent?

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.

Do cells need to be sonicated and/or frozen before treatment with B-PER Reagent?

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.

Are B-PER Reagent components compatible with commercially available 6xHis- and GST-tagged fusion protein purification columns?

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.

Can you provide the shelf-life for the B-PER Bacterial Protein Extraction Reagent?

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.

Can you explain how the B-PER Bacterial Protein Extraction Reagent lyses cells?

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.

The bacteria do not lyse with B-PER reagent. What is the problem?

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.

I lysed my cells with B-PER reagent, but my protein is denatured. How can I get functional protein?

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).

My cells were lysed with B-PER reagent, but now the sample is very "sticky" or viscous. What can I do?

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.

I added enzyme to the B-PER reagent, but my bacteria were not lysed. What can I do?

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.

My bacterial cells were not lysed upon addition of B-PER reagent. How can I increase the lysis efficiency?

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.

I lysed my cells in B-PER reagent, but my protein of interest wasn't solubilized. What can I do?

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.

Which formulation of B-PER bacterial cell lysis reagent is right for me?

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).

What lysis buffers do you offer for bacterial cells?

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

For Research Use Only. Not for use in diagnostic procedures.