Product FAQ

What is the principle underlying the use of Dynabeads magnetic beads for isolation of His-tagged proteins?

Answer

This principle is based on the reversible interaction between various amino acid side chains and immobilized metal ions. Depending on the immobilized metal ion, different side chains can be involved in the adsorption process. Most notably, histidine, cysteine, and tryptophan side chains have been implicated in protein binding to immobilized transition metal ions and zinc. Histidines exhibit highly selective binding to certain metals and have great utility in immobilized metal affinity chromatography (IMAC). Under conditions of physiological pH, histidine binds by sharing electron density of the imidazole nitrogen with the electron-deficient orbitals of transition metals. Although only three histidines may bind transition metals under certain conditions, six histidines reliably bind transition metals in the presence of strong denaturants such as guanidinium. Such protein tags are commonly referred to as 6xHis tags. For isolation of His-tagged proteins, we offer the Dynabeads His-Tag Isolation & Pulldown beads (Cat. Nos. 10103D, 2 mL (40 mg beads/mL) and 10104D, 10 mL (40 mg beads/mL)). These beads have a Cobalt-based Immobilized Metal Affinity Chromatography (IMAC) surface chemistry. The technology is comprised of a tetradentate metal chelator in which four of cobalt's six coordination sites are occupied. The imidazole rings of histidine residues present in a poly histidine peptide chain are able to occupy the two remaining coordination sites, resulting in protein binding.

Answer Id: E6046

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Product FAQ

Can I use BSA as a blocking agent during isolation of His-tagged proteins using Dynabeads His-Tag Isolation and Pulldown magnetic beads?

Answer

BSA would only prevent non-specific adsorption to the surface. Also, if the BSA binds to the chelator, it will block His-tag binding. Instead, we recommend that you use 0.01% Tween 20 detergent instead.

Answer Id: E6052

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Product FAQ

Which reagents are not compatible with His-tagged protein isolation using Dynabeads His-Tag Isolation and Pulldown magnetic beads?

Answer

DTT, DTE, EDTA, and EGTA are not compatible with Dynabeads His-Tag Isolation & Pulldown magnetic beads. You should make sure that these are not present. In addition, we have some evidence that beta-mercaptoethanol and MgCl2 may have an impact on the cobalt as well.

Answer Id: E6053

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Product FAQ

What is the principle underlying the use of Dynabeads magnetic beads for isolation of His-tagged proteins?

Answer

This principle is based on the reversible interaction between various amino acid side chains and immobilized metal ions. Depending on the immobilized metal ion, different side chains can be involved in the adsorption process. Most notably, histidine, cysteine, and tryptophan side chains have been implicated in protein binding to immobilized transition metal ions and zinc. Histidines exhibit highly selective binding to certain metals and have great utility in immobilized metal affinity chromatography (IMAC). At physiological pH, histidine binds by sharing electron density of the imidazole nitrogen with the electron-deficient orbitals of transition metals. Although only three histidines may bind transition metals under certain conditions, six histidines reliably bind transition metals in the presence of strong denaturants such as guanidinium. Such protein affinity tags are commonly referred to as 6xHis tags. For isolation of His-tagged proteins, we offer the Dynabeads His-Tag Isolation & Pulldown beads (Cat. No. 10103D, 2 mL (40 mg beads/mL) and Cat. No. 10104D, 10 mL (40 mg beads/mL)). These beads have a cobalt-based Immobilized Metal Affinity Chromatography (IMAC) surface chemistry. The technology is comprised of a tetradentate metal chelator in which four of cobalt's six coordination sites are occupied. The imidazole rings of histidine residues present in a polyhistidine peptide chain are able to occupy the two remaining coordination sites, resulting in protein binding.

Answer Id: E13043

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Product FAQ

Can the Dynabeads His-Tag Isolation and Pulldown beads be used in mammalian systems?

Answer

Isolation of His-tagged proteins from mammalian cells is problematic due to the endogenous expression of proteins containing polyhistidine tags in mammalian cells. We have not developed a protocol specifically for mammalian systems. To maximize recovery of the recombinant protein (over the endogenous proteins), the expression level of the recombinant protein should be sufficiently high. Given the rapid binding kinetics of the Dynabeads magnetic beads, it is very important to keep the incubation time short (5 to 10 mins) as increasing the incubation time will increase the background. For some proteins, binding efficiency might be affected by which end of the protein the His-tag is on (C or N terminal). In order to reduce background, imidazole (typically 5 mM, but up to a maximum of 20 mM) may be added in the washing solution. Also increasing the linker between the tag and the protein could be considered.

Answer Id: E6050

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Product FAQ

Which product should I choose for my application?

Answer

Which product to choose depends on your ligand, target, sample and application. Both 1 micron (MyOne), 2.8 micron (M-270/M-280) and 4.5 micron (M-450/M-500) Dynabeads magnetic beads can be used. If the sample is very viscous, 4.5 micron beads are recommended. If the target is very fragile, smaller beads are recommended, as they will move more slowly towards the magnet, reducing the risk of squeezing the target. Below is a short product guide showing which beads can be used to immobilize different ligands:

For antibody ligands, use anti-mouse, anti-rat or anti-rabbit coated beads or Dynabeads magnetic beads Protein A/G (2.8 micron beads), Dynabeads magnetic beads M-280 Tosylactivated, Dynabeads magnetic beads M-450 Epoxy or Dynabeads magnetic beads M-500 Subcellular.

For biotinylated ligands, use Dynabeads magnetic beads M-280 Streptavidin. The hydrophilic, negatively charged Dynabeads magnetic beads M-270 Streptavidin or Dynabeads magnetic beads MyOne Streptavidin C1 may also be used.

For protein/peptide ligands (other than antibodies), use Dynabeads magnetic beads M-270 Epoxy, Dynabeads magnetic beads M-270 Amine, Dynabeads magnetic beads M-450 Epoxy.

For histidine affinity-tagged ligands, use Dynabeads magnetic beads His-Tag Isolation and Pulldown beads (1 micron size).

Find more information about Dynabeads magnetic beads at http://www.thermofisher.com/Dynabeads magnetic beads.

Answer Id: E4849

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Product Literature

Brochure: Tools and reagents for recombinant protein purification

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Technical Handbook: Cell and Protein Isolation

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Thermo Scientific Pierce Products for Mass Spectrometry Sample Preparation Handbook

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Surface Activated Dynabeads

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Thermo Scientific Magnetic Bead Technology Brochure

Manual / Product Insert

User Guide: Dynabeads His-Tag Isolation & Pulldown

Version: MAN0017121 Rev. A.0

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Catalog: Protein sample prep and quant for MS

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Catalog: Protein sample prep for MS