Thermo Fisher Scientific SuperSignal West HisProbe Kit User guide

Brand: Thermo Fisher Scientific

Model: SuperSignal West HisProbe Kit

Type: User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

15168 SuperSignal West HisProbe Kit, sufficient for 65 mini-blots or 5000cm2 of membrane

Kit Contents:

HisProbe-HRP, 2mg

SuperSignal West Pico Luminol/Enhancer Solution, 250mL

SuperSignal West Pico Stable Peroxide Solution, 250mL

Blocker™ BSA in TBS (10X), 125mL

BupH™ Tris Buffered Saline Packs, 10 each

Note: The BupH™ Tris Buffered Saline Packs are located under the corrugated cardboard insert.

To access the packs, remove the insert.

Surfact-Amps® 20, 6 × 10mL ampules

Storage: Upon receipt store HisProbe-HRP and Blocker BSA in TBS (10X) and Surfact-Amps 20 at

4°C. All other components may be stored at room temperature. Kit is shipped at ambient temperature.

Introduction

The HisProbe-HRP Technology uses a tridentate metal chelating group capable of binding divalent nickel. The interaction

between divalent nickel and histidines is commonly used to purify polyhistidine-tagged fusion proteins.1-6 Nickel ions can

also be chelated to different ligands. The Thermo Scientific SuperSignal HisProbe Kit combines the HisProbe-HRP

Technology and the SuperSignal West Pico Chemiluminescent Substrate for polyhistidine-tagged fusion protein detection in

Western blotting applications.7-9 HisProbe-HRP is a nickel-activated derivative of horseradish peroxidase (HRP) that is

optimized for direct detection of recombinant polyhistidine-tagged fusion proteins. SuperSignal Substrate is an enhanced

chemiluminescent detection substrate for HRP. This combination of products produces maximum sensitivity that allows for

detection of low expressing clones while maintaining specificity.

A variety of polyhistidine-tagged proteins have been detected with this technology including those expressed in E. coli and

insect Sf9 cells. In comparative experiments with another nickel-activated HRP probe that used a tetradentate chelating

group, HisProbe-HRP exhibited higher specificity to polyhistidine-tagged recombinant proteins and lower background from

non-polyhistidine-tagged proteins.

Furthermore, many monoclonal anti-His antibodies require a specific location of the polyhistidine tag (N- or C-terminus),

primary and secondary antibody incubations, and the presence of specific adjacent amino acid sequences. HisProbe-HRP

requires a simple one-step probe incubation and detects polyhistidine tags independent of location and adjacent sequences.

Detection of polyhistidine-tagged protein by the HisProbe-HRP is, therefore, more versatile than antibody-based detection.

0711.4

15168

SuperSignal® West HisProbe™ Kit

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Material Preparation

Tris Buffered Saline

Tween®-20 Detergent

(TBST)

Dissolve contents of one BupH Tris Buffered Saline Pack in 500mL of ultrapure water and

then add 2.5mL of Surfact-Amps 20. Final composition will be 25mM Tris, 0.15M NaCl, pH

7.6, 0.05% Tween®-20 Detergent.

BSA/TBST

Dilute one volume of Blocker BSA in TBS (10X) into three volumes of TBST. Final

composition will be 25mg bovine serum albumin/mL in TBST.

Note: BSA/TBST can be used for blocking at 10mg BSA/mL TBST if blocking is performed

overnight at 4°C.

HisProbe-HRP Working

Solution Reconstitute vial of HisProbe-HRP with 0.5mL of ultrapure water. Store unused portion at 4°C

for short-term storage or in single-use aliquots at -20°C for long-term storage. Prepare working

solution by diluting 1:5000 in TBST.

SuperSignal West Pico

Substrate Working

Solution

Mix equal volumes of Luminol/Enhancer Solution with Stable Peroxide Solution.

Recommended volume is 0.1mL/cm2 of blot surface.

Note: The Substrate Working Solution is stable for 8 hours at room temperature. Exposure to

the sun or any other intense light can harm the Working Solution. For best results keep the

Working Solution in an amber bottle and avoid prolonged exposure to any intense light. Short-

term exposure to typical laboratory lighting will not harm the Working Solution.

Procedure for Detection of Polyhistidine-Tagged Fusion Proteins

Note: Volumes indicated below are for one 7.5 × 10cm2 blot. Volumes can be adjusted for staining multiple blots or for

different-sized blots.

1. Transfer protein from gel to a nitrocellulose membrane.

Note: Although other membranes can be used, optimization may be required.

2. Place the membrane in a tray and block with 10mL of BSA/TBST for 1 hour at room temperature with shaking.

3. Wash the membrane twice with 15mL of TBST for 10 minutes each.

4. Incubate blot with 10mL of HisProbe-HRP Working Solution for 1 hour with shaking.

5. Wash the membrane four times with 15mL of TBST for 10 minutes each.

6. Incubate blot with 7.5mL of SuperSignal West Pico Substrate Working Solution for 5 minutes.

7. Remove blot from the Substrate Working Solution and place in a membrane sheet protector. Use an absorbent tissue to

remove excess liquid and to carefully press out any bubbles from between the blot and the surface of the membrane

protector.

8. Place the protected membrane in a film cassette with the protein side facing up. Turn off all lights except those

appropriate for film exposure (e.g., a red safelight).

Note: Film must remain dry during exposure. For optimal results, perform the following precautions:

• Make sure excess substrate is removed from the membrane and the membrane protector.

• Use gloves during the entire film-handling process.

• Never place a blot on developed film. There may be chemicals on the film that will reduce signal.

9. Carefully place a piece of film on top of the membrane. A recommended first exposure time is 60 seconds; however,

exposure time can be varied to achieve optimal results. Place the membrane protector containing the blot against film

and expose.

10. Develop the film.

Note: The blot can be re-exposed to film for various times as needed.

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Related Thermo Scientific Products

28376 BupH Tris Buffered Saline Packs, 40 packs

28320 Surfact-Amps 20 (10% Tween®-20 Detergent), 6 × 10mL

37520 Blocker BSA in TBS (10X), 125mL

15165 HisProbe-HRP, 2mg

34090 CL-XPosure™ Film, 5” × 7” sheets, 100 sheets/pkg

21065 Pierce® Background Eliminator Kit, for eliminating background from X-ray film

34080 SuperSignal West Pico Chemiluminescent Substrate, 500mL

88018 Nitrocellulose Membrane, 0.45µm, 33cm × 3m, 1 roll

77010 Nitrocellulose Membrane, 0.45µm, 8 × 12cm, 25/pkg

88025 Nitrocellulose Membrane, 0.45µm, 8 × 8cm, 15/pkg

88600 Western Blotting Filter Paper, 8cm × 10.5cm, 100 sheets

24580 Pierce Reversible Protein Stain Kit for Nitrocellulose Membranes

24585 Pierce Reversible Protein Stain Kit for PVDF Membranes

Cited References

1. Jin, L., et al. (1995). Use of a N,N-bis[carboxymethyl]lysine-modified peroxidase in immunoassays. Anal Biochem 229:54-60.

2. McMahan, S. and Burgess, R.R. (1996). Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the

detection of histidine-tagged proteins immobilized on nitrocellulose. Anal Biochem 236:101-6.

3. Botting, C.H. and Randall, R. E. (1995). Reporter enzyme-nitrilotriacetic acid-nickel conjugates: Reagents for detecting histidine-tagged proteins.

Biotechniques 19(3):362.

4. Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expression and Purification 3:263-81.

5. 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.

6. Hemdan, E., et al. (1989). Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. PNAS 86:1811-

7. L.L. Syklauk, et al. (1997). Nickel chelated to a novel tridentate chelating group binds histidine-tagged fusion proteins: Applications of a nickel-

activated horseradish peroxidase probe and a nickel-activated microtiter plate for assays. Abstract No. 3204, Experimental Biology. New Orleans, LA.

April 6-9, 1997.

8. Chu, R., et al. (1997). SuperSignal HisProbe Western Blotting Kit: Rapid detection of polyhistidine-tagged fusion protein with enhanced specificity,

versatility, and high sensitivity. Previews, 1(3) (Pierce Chemical Co.).

9. Klenk, D., et al. (1997). Detection of polyhistidine-tagged proteins with SuperSignal HisProbe Western Blotting Kit: One-step incubation, high

specificity and sensitivity. 12th International Symposium on Affinity Interactions: Fundamentals and Applications of Biomolecular Recognition.

Kalmar, Sweden. June 15-19, 1997.

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