INSTRUCTIONS

3747 N. Meridian Road

B-PER® 6xHis Fusion

P.O. Box 117
Rockford, IL 61105

Protein Purification Kit

78100 0763.2

Number Description
78100 B-PER® 6xHis Fusion Protein Purification Kit, contains sufficient reagents for five fusion protein
purifications, each from a 250 ml culture
Kit Contents:
B-PER® Bacterial Protein Extraction Reagent, 165 ml, contains 20 mM Tris buffer, pH 7.5, and a
proprietary additive
Nickel Chelated Columns, 5 × 1 ml prepacked columns, contains nickel (Ni2+) chelated iminodiacetic
acid (IDA) that is covalently immobilized to 4% beaded agarose; stored in 0.02 % sodium azide
Wash Buffer 1, 45 ml, contains 35 mM Tris, 150 mM NaCl, 10 mM imidazole, 5% glycerol, pH 7.2,
and 0.5X concentration of proprietary B-PER® Reagent additive
Wash Buffer 2, 60 ml, contains 50 mM Tris, 300 mM NaCl, 25 mM imidazole, 10% glycerol, pH 6.8
Elution Buffer, 45 ml, contains 50 mM Tris, 300 mM NaCl, 200 mM imidazole, 10% glycerol, pH 6.8

Storage: Store B-PER® Reagent at room temperature. Store all other kit components at 4°C. Kit is
shipped at ambient temperature.

Introduction
The B-PER® 6xHis Fusion Protein Purification Kit is for efficient purification of histidine-tagged proteins from bacteria and
baculovirus-infected insect cells. Total soluble protein is first extracted from cells using B-PER® Bacterial Protein Extraction
Reagent, and then the His-tagged protein is purified by immobilized metal affinity chromatography (IMAC) using a pre-
packed column of nickel-chelated agarose. IMAC is also called metal-chelate affinity chromatography (MCAC).
B-PER® Reagent efficiently lyses cells to extract soluble recombinant proteins in a buffer that allows maximum and specific
binding of histidine-tagged proteins to the nickel-chelated agarose. After washing to thoroughly remove nonbound proteins,
the His-tagged proteins are eluted and recovered from the column with an imidazole buffer.
Yield and purity obtained with this kit depends on the expression level, conformation and solubility characteristics of the
recombinant fusion protein (see Important Product Information). Using this kit, Pierce researchers have purified > 10 mg
(> 90% purity) of 6xHis-tagged green fluorescent protein (GFP) from 250 ml overnight bacterial cultures.

Important Product Information

• For best results, perform a small-scale test to estimate the expression level and determine the solubility of the His-tagged
protein: Express protein according to standard protocol, lyse cells with appropriate amount of B-PER® Reagent (see
procedure), centrifuge and analyze supernatant by SDS-PAGE. Only soluble protein extracts that are clarified and have
no particulates can be successfully processed through the affinity column.
• In many cases, B-PER® Reagent will not solubilize proteins that are expressed as inclusion bodies, although the reagent
is effective in purifying inclusion bodies from other cell components so that they can be solubilized with denaturants (see
instructions for B-PER® Reagent, Product No. 78248, which are available from the Pierce web site). Inclusion bodies of
His-tagged proteins that have been solubilized in 8 M urea or 6 M guanidine can be purified with this kit, but denaturant
must be added to the buffers to ensure that the tagged protein remains soluble throughout the procedure.

Warranty: Pierce Biotechnology (hereafter “Pierce”) products are warranted to meet stated product specifications and to conform to label descriptions when stored and used
properly. Unless otherwise stated, this warranty is limited to one year from date of sale when used according to product instructions. Pierce’s sole liability for the product is limited
to replacement of the product or refund of the purchase price. Unless otherwise expressly authorized in writing by Pierce, products are supplied for research use only and are
intended to be used by a technically qualified individual. Pierce’s quality system is certified to ISO 9001. Pierce makes no claim of suitability for use in applications regulated by
FDA. Pierce strives for 100% customer satisfaction. If you are not satisfied with the performance of a Pierce product, please contact Pierce or your local distributor.
• Nickel that is chelated to immobilized iminodiacetic acid (IDA) binds to deprotonated imidazole side-chains of histidine
residues. Nonspecific binding of proteins having isolated individual histidines is minimized by Wash Buffers 1 and 2
because they contain low levels of competing imidazole and are not too alkaline. If an undesirable level of nonspecific
binding occurs, consider increasing Wash Buffer imidazole concentrations and/or decreasing the pH to protonate a
greater proportion of the histidine groups.
• IMAC depends on nickel chelation to both the IDA-agarose and the target histidine tags. Therefore, avoid using protease
inhibitors or other additives that contain chelators, such as EDTA, or strong reducing agents, such as > 5 mM DTT or
2-ME. Chelators will bind and strip nickel from the IDA-agarose, and reducing agents will reduce the metal ion, causing
formation of a brown precipitate.
• Yield and purity obtained with this kit depend on the expression level, conformation and solubility characteristics of the
recombinant fusion protein. For example, histidine tags that are embedded within the protein tertiary structure, as
expressed in the cells and B-PER® Reagent extract, may not be readily accessible for binding the nickel ligands; in such a
case, poor purification yield may result even though protein expression and solubility were good.

Procedure for His-tagged Protein Purification

1. Equilibrate column(s) and buffers to room temperature.
Note: Use either 16 × 150 mm glass tubes or a laboratory stand and clamp to hold the column.
2. Using a 250 ml culture of bacteria (O.D.600 = 1.5-3.0), pellet cells by centrifugation and remove supernatant. If frozen
bacteria are used, thaw to 4°C before starting protein extraction.
3. Suspend the cell pellet in 10 ml of B-PER® Reagent by either vortexing or pipetting up and down until the cell
suspension is homogenous. Gently shake the homogenous mixture at room temperature for 10 minutes.
Note: If desired, add protease inhibitors, such as Halt™ Protease Inhibitor Cocktail, EDTA-Free (Product No. 78410), to
the B-PER® Reagent. Do not use protease inhibitors that contain metal chelators, such as EDTA.
4. Separate soluble from insoluble proteins by centrifugation at 27,000 × g (e.g., 14,000 RPM with Beckman JA17 rotor)
for 15 minutes.
Note: If the extract is so viscous that centrifugation and pipetting are ineffective, add DNAse I (Product No. 89835) to
digest excess DNA, which causes this phenomenon.
5. Remove the supernatant (the protein extract) to a new tube.
Note: Generally, greater than 90% of the soluble proteins are obtained in this supernatant. Performing a second
extraction of the pellet with additional B-PER® Reagent may recover remaining soluble protein.
6. Uncap the Nickel Chelated Column and allow the sodium azide storage solution to drain from the gel bed. The column
will stop flowing when the liquid level reaches the top disc.
7. Prepare the gel bed by adding 10 ml (2 × 5 ml) of B-PER® Reagent and allowing it to flow through the column.
8. Apply up to 10 ml of sample (2 × 5 ml) to the column and allow it to flow through the gel bed.
Note: Collecting and saving separate flow-through fractions obtained in this step and in steps 9 and 10 allows the
efficiency of binding and washing steps to be determined by SDS-PAGE analysis of the fractions.
9. Wash column by adding at least 6 ml (2 × 3 ml) of Wash Buffer 1 and allowing it to flow through the gel bed.
10. Wash column by adding at least 9 ml (3 × 3 ml) of Wash Buffer 2 and allowing it to flow through the gel bed.
11. Elute the 6xHis-tagged protein by adding 6 ml (2 × 3 ml) of Elution Buffer and collecting the fractions that emerge.
Monitor elution by measuring the absorbance at 280 nm or by using the BCA™ Protein Assay (Product No. 23225).
The eluted protein can be directly analyzed by SDS-PAGE. Gel filtration (Zeba™ Desalt Spin Columns) or dialysis
(Slide-A-Lyzer® Dialysis Cassettes) can be used to buffer-exchange and remove excess imidazole.
Note: The Ni-chelated columns can be used at least three times without significant loss of binding capacity. After protein
is completely eluted, wash the gel bed with 2-5 ml of Wash Buffer 1 (Product No. 78110). While solution still remains
above the gel bed, cap the column and store upright at 4°C. Prevent microbial growth during long-term storage by using
Wash Buffer 1 to which sodium azide has been added to 0.01-0.02%. Prevent cross-contamination of samples by
designating a given column to one specific recombinant protein.

In the USA call: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007 • www.piercenet.com
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Troubleshooting
Problem Possible Cause Solution
Low protein yield Poor soluble protein expression Optimize bacterial expression conditions
Fusion protein forms inclusion Alter bacterial growth conditions to minimize inclusion body
bodies formation and maximize soluble protein; typical methods
include harvesting cells earlier in the growth phase after
induction and growing cells at a lower temperature (e.g., 30°C
instead of 37°C). Alternatively, solubilize inclusion bodies and
perform the purification procedure with a compatible denaturant
(e.g., Inclusion Body Solubilization Reagent, Prod. No. 78115).
Insufficient cell lysis and Use recommended amount of B-PER® Reagent and/or freeze
extraction bacteria before performing extraction.
Fusion protein does not bind to Check sequence of the construct to make sure the 6xHis-tag is
the column present and accessible for nickel binding, such as by performing
an ELISA or Western blot using HisProbe™-HRP (Product No.
15165 and 15168)
Poor protein purity Insufficient column washing Wash column additional times with Wash Buffer 1 and 2, or
modify the imidazole concentration and pH of Wash Buffer 2,
as described in the Important Product Information section
Fusion protein interacts with After initial purification, pool fractions that contain the most
other bacterial proteins protein, dialyze to remove imidazole and repeat protocol
Slow column flow Column is overloaded Apply less protein extract onto the column and/or ensure that
the extract is not too viscous or full of particulates

Related Pierce Products

89835 DNase I, 5,000 units
78415 Halt™ Protease Inhibitor Cocktail, EDTA-Free, 1 ml of 100X suspension
78101 Immobilized Nickel Chelated Column, 1 ml pre-packed column
78320 Nickel Chelated Agarose, 8 ml, supplied as 50% slurry in B-PER® Reagent
78110 Wash Buffer 1 for B-PER® 6xHis Kit, 45 ml
78120 Wash Buffer 2 for 6xHis Kits, 60 ml
78130 Elution Buffer for 6xHis Kits, 45 ml
78200 B-PER® GST Fusion Protein Purification Kit
78243, 78248 B-PER® Bacterial Protein Extraction Reagent, 165 ml and 500 ml, respectively
89893 Zeba™ Desalt Spin Columns, 5 × 10 ml, for processing 700-4,000 µl samples
66382 Slide-A-Lyzer® Dialysis Cassette Kit, 10 units and accessories for 0.5-3 ml samples
15165 HisProbe™-HRP, 2 mg
15168 SuperSignal® HisProbe™ Western Blotting Kit
23225 BCA™ Protein Assay Kit

Product References
Dorsey, C.W., et al. (2003).Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis
and transport. Microbiology 149:1227-38.
Kumar, et al. (2001). The autoimmune regulator (AIRE) is a DNA-binding protein. J. Biol. Chem. 276:41357-64.

B-PER® Technology is protected by U.S. Patent # 6,174,704.

SuperSignal® Technology is protected by U.S. Patent # 6,423,662.

Current versions of product instructions are available at www.piercenet.com. For a faxed copy, call 800-874-3723 or contact your local distributor.
©Pierce Biotechnology, Inc., 1/2007. Printed in the USA.

In the USA call: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007 • www.piercenet.com
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