Invitrogen

Penta-His Tag Monoclonal Antibody

Penta-His Tag Antibody in Western Blot (WB)

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Penta-His Tag Antibody (P-21315) in WB

Detection of an oligohistidine fusion protein with the Penta-His mouse monoclonal antibody. Twofold serial dilutions of an Escherichia coli lysate containing overexpressed oligomycin sensitivity-conferring protein (OSCP) fused with a hexahistidine domain were run an SDS-polyacrylamide gel and blotted onto a PVDF membrane. The blot was stained with the SYPRO Ruby protein blot stain (Product # S-11791) to detect the entire protein profile (top). After imaging, the blot was incubated with Penta-His mouse IgG₁ monoclonal antibody (P... View More

Penta-His Tag Antibody in Immunoprecipitation (IP)

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Product Details

P-21315

Applications

Tested Dilution

Publications

Western Blot (WB)

0.1-0.2 µg/mL

View 7 publications 7 publications

Immunohistochemistry (IHC)

1:20-1:50

Immunocytochemistry (ICC/IF)

1:20-1:50

Immunoprecipitation (IP)

View 1 publication 1 publication

Miscellaneous PubMed (MISC)

View 1 publication 1 publication

Product Specifications

Species Reactivity

Tag

Published species

Tag

Host/Isotype

Mouse / IgG1

Class

Monoclonal

Type

Antibody

Immunogen

Histidine Tag (5xHis)

Conjugate

Unconjugated Unconjugated Unconjugated

Form

Lyophilized

Purification

purified

Storage buffer

PBS with PEG, sucrose

Contains

0.08% sodium azide

Storage conditions

4°C

Shipping conditions

Ambient (domestic); Wet ice (international)

AB_2539819

Product Specific Information

Sufficient material is supplied to stain 50-100 minigel blots (8 cm × 10 cm). For immunodetection of oligohistidine fusion proteins on blotting membranes, use the Penta-His antibody at a final concentration of between 0.1 µg/mL and 0.2 µg/mL (1:1,000 to 1:2,000 dilution of the stock solution). Standard buffers and blocking agents, such as 3% BSA (bovine serum albumin) may be used; however, milk powder should not be used, as it reduces sensitivity. Denaturation of the proteins is recommended to ensure exposure of the oligohistidine domain. Detect the Penta-His antibody with an antimouse IgG secondary antibody conjugated to either alkaline phosphatase or horseradish peroxidase. The enzyme activity can then be visualized using a chromogenic, fluorogenic or chemiluminescent substrate.

Upon receipt, the lyophilized antibody should be stored at 4°C. When properly stored, this product is stable for at least one year. To prepare a stock solution, reconstitute the antibody in 0.5 mL of deionized water (dH2O) to a final concentration of 0.2 mg/mL. Store the solution for up to 3 months at 4°C. For storage up to 6 months, divide the solution into single-use aliquots and freeze at -20°C. AVOID REPEATED FREEZING AND THAWING.

Immunohistochemical methods can be used to investigate the location of recombinant proteins expressed in particular cells within tissues or organs, as well as for determination of the subcellular localization of proteins after targeting to particular cell compartments. The Penta-His antibody has been used to detect a hexahistidine aggrecan fusion protein in the perinuclear Golgi and the endoplasmic reticulum of transiently transfected CHO cells. The technique can also be used to localize cellular proteins that interact with hexahistidine fusion proteins. Preparation of the cells or tissue: Fix the tissue with 2% paraformaldehyde and permeabilize with 0.25% Triton™ X-100. This relatively mild fixation protocol best preserves epitopes and best maintains the position of the protein within the cellular structure. Immunodetection for cells and tissues: Before applying the antibody to the tissue, block nonspecific binding sites using 5% bovine serum albumin (BSA). For immunodetection of the oligohistidine fusion protein, use the Penta-His antibody at a final concentration of 410 µg/mL (1:20 to 1:50 dilution of the stock solution). Detect the Penta-His antibody with an anti-mouse IgG secondary antibody conjugated to a fluorescent dye, alkaline phosphatase, or horseradish peroxidase. The fluorescent dye can be seen directly using a fluorescence-based microscope. The enzyme activity can be detected using a chromogenic or fluorogenic substrate.

Target Information

The oligohistidine domain is a Ni2+-binding peptide sequence comprising a string of four to six histidine residues. When the DNA sequence corresponding to the oligohistidine domain is fused in frame with a gene of interest, the resulting fusion protein can be easily purified using a nickel-chelating resin. The Penta-His monoclonal IgG1 antibody provides a sensitive method for specific detection of fusion proteins that have an oligohistidine domain comprising five or six consecutive histidine residues. The antibody does not recognize tetrahistidine domains or domains in which the histidine string is interrupted by another amino acid. The antibody binds to the oligohistidine domain regardless of the surrounding amino acid context and even when the group is partially hidden, although subtle differences in the amino acid context may change the sensitivity limit for a particular fusion protein. This antibody can be used to detect oligohistidine fusion proteins on western blots, dot blots or colony blots, or in tissues.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Product References

Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.

Journal of medicinal chemistry

Poláchová E,Bach K,Heuten E,Stanchev S,Tichá A,Lampe P,Majer P,Langer T,Lemberg MK,Stříšovský K

P-21315 was used in Western Blotting to develop potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation.

Thu Jan 12 00:00:00 UTC 2023

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell-cell adhesion.

The Journal of biological chemistry

Noor SI,Hoffmann M,Rinis N,Bartels MF,Winterhalter PR,Hoelscher C,Hennig R,Himmelreich N,Thiel C,Ruppert T,Rapp E,Strahl S

P-21315 was used in Western Blot to indicate that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.

Mon Aug 30 00:00:00 UTC 2021

A "Drug Sweeping" State of the TriABC Triclosan Efflux Pump from Pseudomonas aeruginosa.

Structure (London, England : 1993)

Fabre L,Ntreh AT,Yazidi A,Leus IV,Weeks JW,Bhattacharyya S,Ruickoldt J,Rouiller I,Zgurskaya HI,Sygusch J

P-21315 was used in Western Blotting to propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux.

Thu Mar 04 00:00:00 UTC 2021

Identification of the Burkholderia pseudomallei bacteriophage ST79 lysis gene cassette.

Journal of applied microbiology

Khakhum N,Yordpratum U,Boonmee A,Tattawasart U,Rodrigues JL,Sermswan RW

Published figure using Penta-His Tag monoclonal antibody (Product # P-21315) in Western Blot

Mon Aug 01 00:00:00 UTC 2016

Structure binding relationship of human surfactant protein D and various lipopolysaccharide inner core structures.

Journal of structural biology

Reinhardt A,Wehle M,Geissner A,Crouch EC,Kang Y,Yang Y,Anish C,Santer M,Seeberger PH

P-21315 was used to study the relationship between lipopolysaccharide inner core structures and the structure binding relationship of human surfactant protein D

Thu Sep 01 00:00:00 UTC 2016

A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth.

Journal of neurochemistry

King MD,Phillips GW,Bignone PA,Hayes NV,Pinder JC,Baines AJ

P-21315 was used in western blot to study the interaction of spectrin with calmodulin to neurite outgrowth through a conserved sequence in calmodulin regulated spectrin-associated protein 1

Sat Feb 01 00:00:00 UTC 2014

Phenotypic characterization of cells participating in transport of prion protein aggregates across the intestinal mucosa of sheep.

Prion

Piercey Åkesson C,Press CM,Tranulis MA,Jeffrey M,Aleksandersen M,Landsverk T,Espenes A

P-21315 was used in western blot to characterize the cells transporting prion protein aggregates across the intestinal mucosa of sheep

Sun Jul 01 00:00:00 UTC 2012

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1.

Nucleic acids research

Felle M,Joppien S,Németh A,Diermeier S,Thalhammer V,Dobner T,Kremmer E,Kappler R,Längst G

Sat Oct 01 00:00:00 UTC 2011