Thermo Fisher Scientific B-PER Direct Bacterial Protein Extraction Kit User guide

Brand: Thermo Fisher Scientific

Model: B-PER Direct Bacterial Protein Extraction Kit

Type: User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

thermofisher.com

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

90080 B-PER Direct Bacterial Protein Extraction Kit, sufficient reagents for five deep-well culture plates

(96-well)

Kit Contents:

B-PER Direct Lysis Buffer, 50 ml

Lysozyme, 0.5 ml supplied at 50 mg/ml in sodium acetate, 50% glycerol; pH 4.7

DNase I, 0.5 ml supplied at 2,500 units/ml in 10 mM Tris•HCl (pH 7.5), 10 mM CaCl2, 10 mM

MgCl2, 50% glycerol

Storage: Upon receipt store enzymes at -20°C and store lysis buffer at ambient temperature. Product

shipped at 4°C.

Introduction

The Thermo Scientific B-PER Direct Bacterial Protein Extraction Kit contains a Tris-buffered detergent concentrate and

enzymes formulated to lyse bacteria (E. coli) directly in culture media. The detergents and enzymes work in conjunction to

gently solubilize proteins and eliminate any particulates in the extract making this process amenable to rapid, high-

throughput protein screening and purification. The B-PER Direct Protocol requires no centrifugation steps and lysates can be

applied directly to affinity purification resins such as Thermo Scientific HisPur Cobalt Resin and immobilized-glutathione.

Important Product Information

• Optimal extraction occurs at ambient temperature. Additional time will be required if performing the extraction at 4°C.

• Use EDTA-free protease inhibitors (Product No. 78425, 78437 or 78439). If the downstream processing includes metal-

chelate affinity purification, avoid adding reagents that contain EDTA.

• Extracts using the B-PER Direct Reagent are compatible at full-strength with the Thermo Scientific Coomassie Plus

(Bradford) Assay (Product No 23236); however, extracts must be diluted 10-fold before performing the Thermo

Scientific Pierce BCA Protein Assay.

Procedure or Protocol for Extraction of Protein from Bacterial Culture

1. Determine the volume of the culture to be lysed.

2. Immediately before use, add 8 µl of lysozyme and 8 µl of DNase I per 1 ml of B-PER Direct Reagent.

3. Add 100 µl of extraction reagent (containing lysozyme and DNase I) for every 1 ml of cell culture and mix by pipetting

up and down.

4. Incubate cells for 15 minutes at room temperature.

5. Apply extract directly to equilibrated affinity resin or process lysates as desired.

Pub. No. MAN0011650

Rev. B.0

Pub Part No. 2162086.1

90080

B-PER™ Direct Bacterial Protein

Extraction Kit

Pierce Biotechnology

PO Box 117

(815) 968-0747

thermofisher.com

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Troubleshooting

Problem

Possible Cause

Solution

Lysate is not clear

Temperature is too low

Warm lysate to ambient temperature or

incubate longer

Protein of interest is expressed in

inclusion bodies

Adjust expression conditions or use an

inclusion body solubilization reagent

Lysozyme has low activity or is inactive

Add more lysozyme to the B-PER Reagent

or purchase new enzyme

Protein quantitation by Pierce

BCA Protein Assay produced

high background

Full-strength extracts are incompatible

with Pierce BCA Protein Assay

Dilute extract 10-fold

Poor protein binding to affinity

resins

Highly acidic culture media exceeds

buffering capacity of B-PER Direct

Reagent

Check and adjust the extract pH before

downstream application

Cannot detect protein of

interest by SDS-PAGE stained

with coomassie

Concentration of protein of interest is low

Silver stain the gel or use undiluted sample

and maximum well volume

Lysate is viscous

DNase I has low activity or is inactive

Add more DNase I to the B-PER Reagent or

purchase new enzyme

Add Mg2+ ions to 2 mM final concentration

Additional Information

A. Please visit the web site for additional information on this product including the following:

• Frequently Asked Questions

• Tech Tip #19: Remove Detergent from Protein Samples

Related Products

90095 HisPur™ 96-well Spin Plates

89807, 89808 Zeba™ 96-well Spin Desalting Plates

90082 Lysozyme, 0.5 ml supplied at 50 mg/ml

90083 DNase I Solution, 0.5 ml supplied at 2,500 U/ml

78115 Inclusion Body Solubilization Reagent, 100 ml

39000 Lane Marker Reducing Sample Buffer, 5 ml

78425 Halt™ Protease Inhibitor Single-Use Cocktail, EDTA-Free

78437 Halt Protease Inhibitor Cocktail, EDTA-Free

XP04200BOX Novex™ Tris-Glycine protein gels (see thermofisher.com/proteingels for a complete listing)

24615 Imperial™ Protein Stain, 1 L, (coomassie R-250)

LC6060 SimplyBlue™ SafeStain

24612 Pierce™ Silver Stain Kit

26619 PageRuler™ Plus Prestained Ladder, 10 to 250 kDa

23227 Pierce BCA Protein Assay Kit

23236 Pierce Coomassie Plus (Bradford) Assay Kit

89802 I-PER™ Insect Cell Protein Extraction Reagent, 250 ml

Pierce Biotechnology

PO Box 117

(815) 968-0747

thermofisher.com

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

78833 NE-PER™ Nuclear and Cytoplasmic Extraction Kit

89826 Mem-PER™ Eukaryotic Membrane Protein Extraction Kit

78501 M-PER™ Mammalian Protein Extraction Reagent, 250 ml

For research use only. Not for use in diagnostic procedures.

This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale,

as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and

workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications

regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the

Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This

warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).

No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular

purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or

refund for the non-conforming Product(s).

There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii)

use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.

Current versions of product instructions are available at thermofisher.com. For a faxed copy, call 800-874-3723 or contact your local distributor.

subsidiaries. Printed in the USA.

Thermo Fisher Scientific B-PER Direct Bacterial Protein Extraction Kit User guide

Thermo Fisher Scientific B-PER Direct Bacterial Protein Extraction Kit User guide

Thermo Fisher Scientific B-PER Direct Bacterial Protein Extraction Kit User guide

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