Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

Specialty Assays – Histidine-tagged Proteins
Histidine-tagged Protein Detection
Thermo Scientific HisProbe-HRP Western blotting probe takes
advantage of the affinity of histidine for the Ni 2+ cation.
A. B.
HisProbe-HRP is a nickel (Ni 2+ )-activated derivative of horseradish
peroxidase (HRP). This product has been optimized for
direct detection of recombinant histidine-tagged proteins and
other histidine-rich proteins. The active ligand is a tridentate
chelator that allows Ni 2+ to be bound in active form for subsequent
interaction and detection of target molecules. The active
chelator has similar binding capabilities to that reported for
iminodiacetic acid, which has long been used for immobilized
metal affinity chromatography (IMAC).
Highlights:
• Yields lower background than anti-histidine antibodies
• Pierce HRP is a high-activity enzyme
• Stripping and reprobing is possible
• HisProbe-HRP (Ni 2+ ) can be used for detection of
histidine-tagged proteins
Substrate
Signal
Ni 2+
Ni 2+ HRP Ni 2+
HisHisHisHisHisHis
6His
Protein
Detection of histidine-tagged fusion proteins with Thermo Scientific
HisProbe-HRP.
Ordering Information
Product Description Pkg. Size
15165 HisProbe-HRP 1mg
15168 SuperSignal ® West Pico HisProbe Kit
Includes: HisProbe-HRP
SuperSignal West Pico
Chemiluminescent Substrate
BSA in TBS (10X)
BupH Tris Buffered Saline Packs
Surfact-Amps ® 20 (10%)
Kit
2mg
500mL
1 x 125mL
10 x 500mL
6 x 10 ampules
Panel A using Thermo Scientific HisProbe-HRP shows high specific binding
and low background.
Panel B using anti-polyHis failed to recognize two of the three fusion proteins.
References
Adler, J. and Bibi, E. (2004). Determinants of substrate recognition by the Escherichia
coli multidrug transporter MdfA identified on both sides of the membrane. J. Biol.
Chem. 279, 8957-8965.
Adler, J. and Bibi, E. (2005). Promiscuity in the geometry of electrostatic interactions
between the Escherichia coli multidrug resistance transporter MdfA and cationic
substrates. J. Biol. Chem. 280, 2721-2729.
Boulant, S., et al. (2003). Unusual multiple recoding events leading to alternative forms
of hepatitis C virus core protein from genotype 1b. J. Biol. Chem. 278, 45785-45792.
Kanaya, E., et al. (2001). Zinc release from the CH 2 C 6 zinc finger domain of filamentous
flower protein from Arabidopsis thaliana induces self-assembly. J. Biol. Chem. 276,
7383-7390.
Robalino, J., et al. (2004). Two zebrafish eIF4E family members are differentially
expressed and functionally divergent. J. Biol. Chem. 279, 10532-10541.
Robichon, C., et al. (2005). Depletion of apolipoprotein N-acyltransferase causes
mislocalization of outer membrane lipoproteins in Escherichia coli. J. Biol. Chem. 280,
974-983.
Segawa, H., et al. (2005). Reconstitution of GDP-mannose transport activity with purified
Leishmania LPG2 protein in liposomes. J. Biol. Chem. 280, 2028-2035.
Sundberg-Smith, L., et al. (2005). Adhesion stimulates direct PAK1/ERK2 association and
leads to ERK-dependent PAK1 Thr212 phosphorylation. J. Biol. Chem. 280, 2055-2064.
Wagner, C., et al. (2005). Dimerization of NO-sensitive guanylyl cyclase requires the α1
N terminus. J. Biol. Chem. 280, 17687-17693.
Wann, E., et al. (2000). The fibronectin-binding MSCRAMM FnbpA of Staphylococcus
aureus is a bifunctional protein that also binds to fibrinogen. J. Biol. Chem. 275,
13863-13871.
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For more information, or to download product instructions, visit www.thermoscientific.com/pierce