INSTRUCTIONS

HisProbe-HRP
15165
Number
15165 0674.4

Description
HisProbe-HRP, 2mg, supplied lyophilized in 0.1M MES, pH 5.5 Storage: Upon receipt store product at 4C. Product is shipped at ambient temperature.

Introduction
The Thermo Scientific HisProbe-HRP is a nickel (Ni2+) activated derivative of horseradish peroxidase (HRP) used for direct detection of recombinant poly-histidine-tagged fusion proteins and other histidine-rich proteins. Isolated from horseradish root, HRP is a high activity enzyme label used to detect small amounts of antibodies, protein, peptides, DNA and RNA in ELISA, immunohistochemistry and Western blotting applications. Nickel is used for binding interactions with histidines and is the basis for several approaches for recombinant histidine-tagged protein purification and detection strategies.1-3 Binding can occur even in chaotropic agents, such as 8M urea or 6M guanidineHCl, allowing detection of insoluble proteins. Nickel also binds IgG antibodies through the histidine repeat in the Fc region. Although crosslinking HRP to the target molecule can create specific probes, and indirect HRP probes are often commercially available, these systems require additional processing steps. HisProbe-HRP allows a simple method for directly detecting histidine-rich proteins.

Important Product Information

Occasionally, folding may prevent detection of histidine-tagged proteins. Treatment with 3M thiocyanate may expose buried histidines.1 A variety of solutes are inert with respect to metal interactions on macromolecules including nonionic detergents, urea, ethylene glycol and dimethylsulfoxide.4 Sodium chloride may enhance or decrease metal affinity interactions. Imidazole is used as an eluent for interactions involving nickel, cobalt, zinc, and copper. Avoid EDTA and other chelators, as they will interfere with binding. Also avoid reducing agents such as mercaptoethanol. Metal binding to the target molecule may be related to topology, microenvironment, reversible reactions, pH, ionic strength, and other factors.5 There appears to be an inverse correlation between the pK values of the histidine and avidity of protein for metal chelates.6

Example ELISA Procedure for Detection of Poly-histidine-containing Proteins

A. Materials Required Coating Buffer: 0.1M sodium bicarbonate buffer; pH 9.6 (Product No. 28382) Wash Buffer: Phosphate-buffered saline containing (PBS) 0.1M sodium phosphate, 0.15M NaCl; pH 7.2 (Product No. 28372). Add Tween-20 Detergent (Product No. 28320) to a final concentration of 0.05%. Blocking Buffer: Prepare 1% BSA by diluting Blocker BSA (10) in PBS (Product No. 37525). Other blocking buffers may be used; however, optimization may be required. HisProbe-HRP Working Solution: Reconstitute HisProbe-HRP with ultrapure water for a final concentration of 5mg/mL. The stock solution may be stored at 4C or aliquoted into single-use volumes and frozen for long-term storage. Prepare Working Solution by diluting the stock solution to 1 g/mL with Wash Buffer. Thermo Scientific 1-Step Turbo TMB ELISA Substrate (Product No. 34022) 1N Sulfuric acid (H2SO4)
Pierce Biotechnology 3747 N. Meridian Road PO Box 117 Rockford, lL 61105 USA (815) 968-0747 (815) 968-7316 fax www.thermoscientific.com/pierce

B. Method 1. 2. 3. 4. 5. 6. Coat 100L of histidine-containing protein(s) by adding 5-20g/mL in Coating Buffer to plate wells. Incubate overnight at 4C or for two hours at room temperature. Block nonspecific binding sites by adding 200L of Blocking Buffer to each well. Incubate for 30 minutes at 37C. Wash plate three times with 200L of Wash Buffer. Add 100L of HisProbe-HRP Working Solution to each well and incubate for 15 minutes at room temperature. Wash plate four times with 200L of Wash Buffer. Add 100L of 1-Step Turbo-TMB ELISA and allow color to develop for 5-30 minutes at room temperature. Stop substrate reaction by adding 50L of 1N sulfuric acid. Measure absorbance at 450nm.

Example Western Blot Procedure for Detection of Poly-histidine-containing Proteins

A. Materials Required TBST: Tris-buffered saline containing 25mM Tris, 0.15M NaCl; pH 7.2 (Product No. 28379). Add Tween-20 Detergent (Product No. 28320) to a final concentration of 0.05%. Blocking Buffer: Dilute one volume Blocker BSA in TBS (Product No. 37520) into three volumes of TBST. Final composition will be 25mg BSA/mL TBST. HisProbe-HRP Working Solution: Reconstitute HisProbe-HRP with 0.5mL of ultrapure water for a final concentration of 4mg/mL. The stock solution may be stored at 4C after reconstitution or aliquoted into single-use volumes and frozen for long-term storage. Prepare Working Solution by diluting the stock solution 1:5000 with Blocking Buffer. Nitrocellulose membrane containing transferred protein. Although other membranes types may be used, optimization may be required. Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate Working Solution (Product No. 34080). Mix equal volumes of Luminol/Enhancer Solution with Stable Peroxide Buffer. Use a volume of 0.125mL/cm2 of blot surface.

B. Method Note: Volumes indicated are for one 7.5 10cm2 blot and may be adjusted for other blot sizes or multiple blots. 1. 2. 3. 4. 5. 6. 7. Place membrane in a tray and block nonspecific binding sites with 10mL of Blocking Buffer for 1 hour at room temperature with shaking. Wash membrane twice with 15mL of TBST for 10 minutes each. Incubate blot with 10mL of HisProbe-HRP Working Solution for 1 hour with shaking. Wash membrane four times with 15mL of TBST for 10 minutes each. Incubate blot with 10mL of SuperSignal Working Solution for 5 minutes. Remove blot from the SuperSignal Working Solution and place in a sheet protector or clear plastic wrap. Place the protected blot against film and expose. A recommended first exposure is 30 seconds. The blot can be reexposed to film for various times as needed.

Pierce Biotechnology 3747 N. Meridian Road

PO Box 117 Rockford, lL 61105 USA

(815) 968-0747 (815) 968-7316 fax

www.thermoscientific.com/pierce

Related Thermo Scientific Products

Please consult our catalog for a complete listing of products including buffers, transfer membranes and activated microplates. Colorimetric HRP substrates for ELISA (products are listed from most sensitive) 34028 34021 34022 34006 34024 37615 34065 34000 34018 34075 34080 References
1. 2. 3. 4. 5. 6. Jin, L., et al. (1995). Use of a-N,N-bis[carboxymethyl]lysine-modified peroxidase in immunoassays. Anal Biochem 229:54-60. McMahan, S. and Burgess, R. R. (1996). Single-step synthesis and chracterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose. Anal Biochem 236:101-6. Botting, C., H. and Randall, R. E. (1995). Reporter enzyme-nitrilotriacetic acid-nickel conjugates: reagents for detecting histidine-tagged proteins. Biotechniques 19(3):362. Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expr Purif 3:263-81. Belew, M. and Porath, J. (1990). Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density, and salt concentration on the retention of peptides. J Chrom 516:333-54. Hemdan, E., et al. (1989). Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc Natl Acad Sci USA 86:1811-15.

1-Step Ultra TMB-ELISA, 250mL TMB Substrate Kit 1-Step Turbo TMB, 250mL OPD Tablets 1-Step Slow TMB, 250mL 1-StepABTS, 250mL DAB Metal Enhanced Substrate Kit CN/DAB Substrate Kit 1-Step TMB-Blotting, 250mL SuperSignal West Dura Extended Duration Substrate, 100mL SuperSignal West Pico Chemiluminescent Substrate, 500mL

Peroxidase substrates for blotting and histochemistry

Chemiluminescent HRP substrates for blotting

Tween is a trademark of Croda International Plc. 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. Buyers 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 product instructions are available at www.thermoscientific.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor. 2011 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.

Pierce Biotechnology 3747 N. Meridian Road

PO Box 117 Rockford, lL 61105 USA

(815) 968-0747 (815) 968-7316 fax

www.thermoscientific.com/pierce