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4.1 Affinity-based Protein Purification 45
HaloTag ®^ Fusion Proteins
HaloTag ®^ Protein Purification System (E. coli) 48 HaloTag ®^ Mammalian Protein Purification and Detection Systems 49
His-tagged Proteins
HisLink ™^ Protein Purification Resin 51 HisLink ™^ Spin Protein Purification System 52 HisLink ™^ 96 Protein Purification System 53
Biotinylated Proteins
SoftLink ™^ Soft Release Avidin Resin 54 PinPoint ™^ Xa Protein Purification System (Production and Purification) 55
4.2 Magnetic Affinity-based Purification
and Pull-Down Strategies 56
GST-tagged Proteins
MagneGST ™^ Protein Purification System 57
HaloTag ®^ Fusion Proteins
Magne ™^ HaloTag®^ Beads 59
His-tagged Proteins
MagneHis ™^ Protein Purification System 60 Maxwell ®^ 16 Polyhistidine Protein Purification Kit 61
Magnetic Stands and Spacers
Magnetic Stands and Spacers 62
Protein Purifi cation
Protein purification is fundamental to the study of protein function and involves a series of processes
to express, enrich and purify a protein of interest from
a complex mixture such as cell lysate. The fastest and most powerful method for this purpose is affinity
purification. During the purification of recombinant proteins it is convenient to use affinity tags such as
HaloTag, GST- or His-tag. By introducing a cleavage- site between the protein fusion tag and the protein
sequence, the tag can be enzymatically removed after the purification procedure.
In this chapter different affinity tags will be introduced as well as the corresponding resins and magnetic
supports that can be used for low- to high-
throughput protein purification.
Protein Purifi cation
46 Discover Reliable Tools for Protein Analysis46 Discover Reliable Tools for Protein Analysis
4
Biotin Tags
The power of the streptavidin-biotin interaction is used in a number of applications, including the detection of various biomolecules. Bacterial streptavidin and its analog chicken avidin have a very high binding affinity for biotin (vitamin H). In fact, this is one of the strongest non-covalent interactions known in biology. The strength of the binding is a major drawback for its use as a puri- fication technique, since the elution conditions would have to be so harsh that they would destroy the purified protein.
Nevertheless, there are several ways to circumvent this strong binding and harness the power of the streptav- idin-biotin interaction for the purification of recombinant proteins. One strategy is to fuse the protein of interest (POI) to a peptide sequence that is biotinylated in vivo. The biotinylation peptide sequence is fused to a protease cleavage site allowing a gentle release of the protein of interest by protease digestion. Other strate- gies are based on biotin-mimicking peptides such as Strep Tag, which binds to native and modified versions of streptavidin and avidin, enabling successful protein elution under mild conditions (e.g., competitive elution with D-desthiobiotin).
Glutathione-S-Transferase (GST)
The use of the affinity tag glutathione-S-transferase (GST) is based on the strong affinity of GST for immo- bilized glutathione-covered matrices. Glutathione-S- transferases are a family of multifunctional cytosolic proteins that are present endogenously in eukaryotic organisms but normally not found in bacteria. As such, GST-based purification is not recommended for eukaryotic systems (e.g., insect, mammalian). The 26kDa GST affinity tag enhances the solubility of many eukaryotic proteins expressed in bacteria. After capture and washing, the tagged protein is eluted with soluble glutathione.
HaloTag®^ Protein Tag
Protein fusion tags are frequently used to aid in expression of suitable levels of soluble protein as well as for purification. The HaloTag®^ fusion protein is engineered to enhance expression and solubility of recombinant proteins in E. coli , as well as to provide superb recovery of even low- expressing proteins, such as in mammalian expression systems. HaloTag®^ technology is based on the formation of a covalent bond between a protein fusion tag (HaloTag®) and synthetic chemical ligands. By interchanging different synthetic ligands researchers ultimately control the function and properties of the HaloTag®^ fusion protein. The HaloTag® system is designed to provide broad experimental flexibility and superb performance in both cell-based and biochemical assays such as protein purification, without switching fusion tags, or recloning. Available HaloTag®^ ligands include magnetic and nonmagnetic beads, glass slides, cell- permeable and impermeable fluorescent ligands, biotin, and reactive ligands to enable researchers to build their own custom HaloTag®^ ligands and surfaces. (Figure 4.1). For protein purification HaloTag®^ Technology is compatible with many protein expression systems and can be applied to proteins expressed in E. coli , mammalian cells and cell- free systems. HaloTag®^ protein is a good affinity tag for puri- fication and also a good solubility tag. The lack of an endog- enous equivalent of the HaloTag®^ protein in mammalian and most prokaryotic cells minimizes the chances of nonspecific interactions. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags, and enables efficient capture even at low expression levels.
47 Discover Reliable Tools for Protein Analysis47 Discover Reliable Tools for Protein Analysis
4
12148MB
HaloTag ®
HaloTag®^ Ligand (Chloroalkane + Functional Group)
Covalent Bond
TEV
cleavage site
Covalent bond between HaloTag ®^ protein and HaloTag ®^ ligand (chloroalkane + functional group).
+ Cl– O O
O O
Cl –
POI
HaloTag ® POI
Fluorescent Ligands: for Cellular Imaging, Detection and Protein-Interaction Studies (BRET). They come in many different colors, as cell permeable ligands and as non-permeable ligands.
Non-fluorescent Ligands: for Protein Detection, e.g., Biotin; PEG-Biotin.
Surface Ligands: for Protein Purification and Protein-Interaction Studies (Pull-downs), e.g., Magnetic Beads; Resins; Arrays.
Reactive Ligands: to attach functional group of choice, e.g., Positron Emission Tomography (PET) ligands, magnetic resonance reagents.
Figure 4.1. Schematic of HaloTag®^ technology consisting of the HaloTag®^ protein fused to a protein of interest (POI) and a selection of synthetic HaloTag®^ ligands carrying different functional groups. HaloTag®^ ligands specifically and covalently bind to the HaloTag®^ protein fusions.
4
HaloTag
® Mammalian Protein Purifi cation and
Detection Systems
Quick purification of HaloTag®^ fusion proteins from mammalian cell culture lysates
and supernatant.
Description
The HaloTag®^ Mammalian Protein Purification System is an optimized kit for rapid purification of HaloTag® fusion proteins from mammalian cell culture lysates and cell culture supernatants. An optimized TEV protease recognition site within the interconnecting polypeptide separates the HaloTag®^ protein and the fusion partner. The kit contains HaloLink™^ Resin, HaloTEV Protease, 50X Protease Inhibitor Cocktail as well as Spin Columns. The covalent binding of HaloTag®^ fusion proteins, coupled with the low nonspecific binding of the HaloLink™^ Resin, provides superior purity and recovery of recombinant proteins from cultured mammalian cells, even at low expression levels. The HaloTag®^ Mammalian Protein Detection and Purification System also contains a fluores- cent ligand (TMRDirect™^ Ligand) for the easy detection of HaloTag®^ fusion proteins by in-gel imaging, flow cytometry or microscopy applications. The simple-to-use fluorescent detection of HaloTag®^ fusion proteins facilitates rapid opti- mization of expression and purification conditions.
Principle
The protein of interest (POI), when fused to the HaloTag ®^ protein, will covalently bind to the HaloLink™ Resin (Figure 4.2). The covalent nature of the linkage enables stringent and lengthy wash conditions without concern of leaching the protein of interest off the resin. Post wash the POI is eluted by proteolytic cleavage with HaloTEV Protease. Since the HaloTEV Protease is fused to HaloTag®^ protein, the cleavage step and protease capture can be performed in a single step that separates the POI from the HaloTag fusion tag and the HaloTEV Protease.
9532MB
Covalently bind the HaloTag®^ fusion protein (POI) to HaloLink™^ Resin.
Release POI using HaloTEV Protease, which will remain covalently attached to the HaloLink™^ Resin.
Recover purified protein of interest.
HaloTEV Protease HaloTag®^ protein Protein of Interest
HaloLink™ Resin
HaloLink™ Resin
POI POI
POI
POI
Figure 4.2. Schematic of the purification steps used with the HaloTag® Mammalian Protein Purification System.
Affi nity-based Protein Purification
Features and Benefits
- High Purity: Covalent capture allows extensive and/or stringent washes without loss of bound protein, result- ing in very low (<0.1%) nonspecific binding and a highly pure protein.
- High Protein Recovery: Rapid covalent capture. Recovery is highly efficient, commonly >75%.
- High Yield: >7mg HaloTag®^ fusion protein per ml of HaloLink ™^ Resin.
- Easily Scalable: From 1ml up to 1L of mammalian cell culture.
- Easy Detection: Fluorescent HaloTag®^ ligands facilitate monitoring and optimization of the protein expression and purification procedure.
4
Fluorescent labeling of HaloTag®^ fusion proteins with the HaloTag ®^ TMRDirect™^ Ligand provides a rapid and convenient method to optimize protein expression and to monitor purification efficiency. HaloTag®^ protein fusions are briefly incubated with HaloTag®^ TMRDirect™^ Ligand prior to SDS-PAGE (Figure 4.3). After electrophoresis the proteins can be imaged without additional processing using a either a fluorescence gel scanner, such as GE Typhoon, or BIORAD ChemiDoc™^ MP system.
Affi nity-based Protein Purification
Ordering Information HaloTag®^ Mammalian Protein Purification System (Cat.# G6790) HaloTag®^ Mammalian Protein Detection and Purification System (Cat.# G6795)
Figure 4.3 Schematic diagram of the fluorescent labeling of HaloTag® fusion protein with the HaloTag®^ TMRDirect™^ Ligand.
9688MA
Dilute HaloTDilute HaloTDilute HaloTagag ®^ TMRDirect™ Ligand to make a 50μM working solution.
Combine lysate, HaloTCombine lysate, HaloTCombine lysate, HaloTagag ®^ Protein Purification Buffer and HaloTfer and HaloTfer and HaloTagag ® TMRDirect™ Ligand.
Add 4X SDS gel loading buffer.
Load samples onto SDS-polyacrylamide gel.
Scan gel (532Ex, 580Em).
Incubate at room temperature for 15 minutes.
Incubate at 70°C for 3 minutes.
References
Mammalian Cells Ohana, R. et al. (2011) HaloTag-based purification of functional human kinases from mammalian cells. Protein Expr. Purif. 76 (2), 154–64. Sun, X. et al. (2013) Hsp90 inhibitor 17-DMAG decreases expression of conserved herpesvirus protein kinases and reduces virus production in Epstein-Barr virus-infected cells. J. Virol. 87 (18), 10126–38. Ryu MJ, (2012) Oncogenic Kras expression in postmitotic neurons leads to S100A8-S100A9 protein overexpression and gliosis. J. Biol. Chem. 287 (27), 22948–58. Plant Cells Lang, C. (2006) Purification from Plant. HaloTag: a new versatile reporter gene system in plant cells. J. Exp. Bot. 57 (12), 2985–92. Yeast Kardon, J.R. (2009) Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin. Proc. Natl. Acad. Sci. USA. 106 (14), 5669–74.
Additional Information
Depending on the orientation of fusion constructs, 6 amino acids for N-terminal fusion constructs or 13 amino acids for C-terminal fusion constructs will remain on the POI after HaloTEV cleavage.
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Affi nity-based Protein Purification
HisLink
™ Spin Protein Purification System
Purification of polyhistidine (His)-tagged proteins in small volumes from bacterial cells.
Description
The HisLink™^ Spin Protein Purification System provides a simple and fast system for purifying overexpressed His-tagged proteins from a 700μl sample of E. coli cell culture, using either a centrifuge- or vacuum-based method. The system contains cell lysis buffer, HisLink™^ Resin, DNase I, Buffers, Collection Tubes and Spin Columns.
Ordering Information HisLink™^ Spin Protein Purification System (Cat.# V1320)
Principle
Protein can be purified directly from culture medium containing bacterial cells expressing a polyhistidine-tagged protein. The bacterial cells are lysed using FastBreak™^ Cell Lysis Reagent, followed immediately by addition of HisLink™ Protein Purification Resin to the culture. Addition of these reagents results in simultaneous bacterial cell lysis and binding of the polyhistidine-tagged proteins. The samples then are transferred to a Spin Column where unbound protein is removed while the affinity resin is washed, and the target protein is recovered by elution. This system requires the use of a tabletop centrifuge or vacuum manifold. A schematic diagram of protein purification using the HisLink ™^ Spin System is shown in Figure 4..
Features and Benefits
- Simple: No cell culture preparation steps (no preclearing) required.
- Quick: No lengthy lysozyme incubations required to lyse cells.
- Efficient: Binding capacity of 1mg of polyhistidine- tagged protein per spin column.
Reference
Engel, L. et al. (2006) HisLink™^ Spin Protein Purification System: Maximum Versatility in a Small Package. Promega Notes 93 , 2-4.
Lyse cells and bind pr the resin.otein to
Transfer contents to Spin Column in a Collection TCollection TCollection Tube orube or Vacuum Adapter.
Spin Column Spin Column
V Adapteracuum Collection TCollection TCollection Tubeube
Centrifuge or apply vacuum for 5 seconds.
Centrifuge or apply vacuum for 5 seconds.
Centrifuge 1 minute.
Add HisLink™ Binding/Wash Buffer.
Place Spin Column into a new 1.5ml micr tube. Add 200μl of HisLink™ocentrifuge Elution Buffer.
Repeat wash steps.
Figure 4.4. Schematic of polyhistidine-tagged protein purification using the HisLink™^ Spin Protein Purification System.
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HisLink
™ 96 Protein Purification System
Purification of polyhistidine (His)-tagged proteins from bacterial and mammalian
cells using a vacuum-based format.
Description
The HisLink™^ 96 Protein Purification System provides a simple, and quick method of high-throughput purification of polyhi- stidine- or HQ-tagged overexpressed proteins from E. coli using a vacuum-based method. The system is designed to purify expressed polyhistidine-tagged proteins directly from deep-well 96-well culture plates. The HisLink™^ System is amenable to manual or automated methods, such as the Beckman Coulter Biomek®^ 2000 or FX for high-throughput applications. The System contains cell lysis buffer, HisLink™^ Resin, DNase I, Buffers and HisLink™^ 96 Filtration & Collection Plates.
Principle
In preparation for protein purification, bacterial cells expressing a polyhistidine-tagged protein are lysed in culture using the provided FastBreak™^ Cell Lysis Reagent. The HisLink™^ Resin is added to the lysate and mixed; the polyhistidine-tagged proteins bind within a few minutes. Transfer the samples to a Filtration Plate, wash the resin to remove contaminants, and recover the target protein by elution (Figure 4.5).
Features and Benefits
- Simple: No centrifugation required—lysis buffer is added directly to cells in culture medium.
- Quick: No long lysozyme incubations are required for cell lysis.
- Versatile: Perform purification manually or on an automated platform.
- Efficient: Binding capacity of 1mg of polyhistidine- tagged protein per well.
Additional Information
Note: This system requires the use of the Vac-Man®^96 Vacuum Manifold or compatible vacuum manifold.
Reference
Engel, L. et al. (2005) HisLink™^ 96 Protein Purification System: Fast Purification of Polyhistidine-Tagged Proteins. Promega Notes 90 , 15–18.
4927M
B
Add FastBreak™ Cell Lysis/DNase I Solution and HisLink™ Resin to 1ml cell cultures in deep-well plate.
TTTransfer lysates andransfer lysates and HisLink™ Resin to filtration plate and apply vacuum. Filtration Plate
Elution Plate
Overnight cultures
Wash.
Elute protein.
Purified polyhistidine- or HQ-tagged proteins.
Figure 4.5. Flow diagram of polyhistidine-tagged protein purification using the HisLink ™^ 96 Protein Purification System.
Ordering Information HisLink™^ 96 Protein Purification System (Cat.# V3680, V3681)
Affi nity-based Protein Purification
4
PinPoint
™ Xa Protein Purifi cation System
Production and purification of fusion proteins that are biotinylated in E. coli.
- Wash.
- Elute with free biotin.
PinPoint™ Expression Vector
- Express fusion protein in E. coli.
- Lyse cells, digest with nuclease.
- Pellet cell debris. Biotinylated fusion protein in crude cell supernatant
Detection using avidin reagents
Proteolytic removal of biotinylated tag peptide
0367MC03_3A
Bind to SoftLink™ Soft Release Avidin Resin.
Figure 4.6. Schematic diagram of recombinant protein expression and purification using the PinPoint™^ Xa Protein Purification System.
Description and Principle
The PinPoint™^ Xa Protein Purification System is designed for the production and purification of fusion proteins that are biotinylated at the N-terminus in vivo. The DNA coding for the protein of interest is cloned into a PinPoint™^ Vector downstream of a sequence encoding a 14kDa peptide that becomes biotinylated in vivo. Biotinylated fusion proteins are produced in E. coli (JM109 and HB101 E. coli bacterial strains, or other strains without the birA mutation) and are affinity-purified using the SoftLink™^ Soft Release Avidin Resin. The PinPoint™^ Vectors feature the encoded endoproteinase Factor Xa, for proteolytic removal of the biotinylated peptide post-purification. The system contains 3 cloning vectors in multiple combinations of sense reading frames, an avidin- conjugated resin, Streptavidin-Alkaline Phosphatase, a purification column and biotin. The PinPoint™^ Xa Control Vector contains the chloramphenicol acetyl- transferase (CAT) gene and is provided as a means of monitoring protein expression, purification and processing conditions. The system generally yields 1–5mg of protein per liter of E. coli culture.
Features and Benefits
- In vivo Biotinylation Tag: Allows purification of fusion proteins labeled with biotin.
- Easy-to-Use: Purification of biotinylated proteins with the SoftLink™^ Resin can be performed by col- umn or batch purification.
- Flexible: PinPoint™^ Vectors are supplied for all reading frames.
- Gentle Release Conditions: SoftLink™^ Resin allows release of the fusion protein under nondenaturing conditions. Ordering Information PinPoint™^ Xa Protein Purification System (Cat.# V2020)
Affi nity-based Protein Purification
56 Discover Reliable Tools for Protein Analysis
4
56 Discover Reliable Tools for Protein Analysis
The use of magnetic beads for protein purification/pull- down provides a rapid and efficient method to extract and capture recombinant proteins from cell lysates and cell culture supernatant. Virtually all affinity chemistries discussed previously can be transferred on magnetic beads. There are many advantages of a magnetic approach:
- Easily process very small sample volumes. Proteins from volumes as low as 20μl can be processed with good efficiency and minimal target protein losses.
- Simple and cost effective approach that does not require extensive plumbing, chromatography instrumentation or centrifugation.
- Protein samples can be eluted into a minimal volume resulting in higher concentration of purified protein. This can be used for protein concentration purposes.
- Faster washing and elution steps because no centrifugation steps are required.
- More readily adapted for high-throughput applications on automated liquid-handling robots (Figure 4.7).
- Magnetic approach excels at parallel sample processing, where multiple samples need to be processed as fast as possible.
All previously discussed affinity chemistries (His, HaloTag ®^ GST) are available for protein capture in a magnetic bead format and are amenable to both protein purification and protein pull-down applications.
4.2 Magnetic Affinity-based Purification and
Pull-down Strategies
OVERVIEW
Figure 4.7. Magnetized beads in an Eppendorf tube during protein purification from small (μl) sample volumes (Panel A). Tecan automated liquid handler processing magnetic beads during high-throughput purification (Panel B).
10543TB
A.
B.
4
MPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMP
6544MB
TNT®-based Cell-Free Expression of Prey Protein
Cell Lysis
Bait expressed as GST Fusion in E.coli
Bind, Wash and Capture on MagneGST™^ Glutathione Particles
Mix and Allow Interaction to Occur
Gel Electrophoresis Followed By Detection (Western Blotting or Autoradiography)
Detect Prey Protein
Wash and Elute Complex
Plasmid Containing Protein Coding Sequence for Prey Protein
RNA Promoter
Prey SequenceSequenceSequence
GGGGGGGGGGGGGGGGGG SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS TTTTTTTTTTTTTTTT
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
B - Bait Protein P - Prey Protein
GST - Glutathione S-transferase
MP - Magnetic Particle
GGGGGGGGGGGGGGGGGG SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS TTTTTTTTTTTTTTTT
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
GGGGGGGGGGGGG SSSSSSSSSSSSSSSSSSSSSS TTTTTTTTTTTT
MPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMPMP BBBBBBBBBBBBBBBBBBBBBBBBBBBB PPPPPPPPPPPPPPPPPPPPPPPP
PPPPPPPPPPPPPPPPPPPPPPPPPPP
GGGGGGGGGGGGGGGG SSSSSSSSSSSSSSSSSSSSSSS TTTTTTTTTTTT
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB PPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPP
Figure 4.8. Schematic diagram of MagneGST™^ Protein Purification in combination with protein pull-down.
Magnetic Affi nity-based Protein Purifi cation
4
Magne
™ HaloTag
® Beads
Manual or automated high-throughput protein purification and pull-down of HaloTag ®^ fusion proteins from E. coli and mammalian cell cultures, at input
volumes from 20μl to 50ml.
Ordering Information Magne™^ HaloTag®^ Beads (Cat.# G7281, G7282)
Figure 4.9 Schematic diagram of the HaloTag®^ fusion purification protocol using Magne™^ HaloTag®^ Beads.
10441MA
Equilibrate Magne™ HaloTag®^ Beads.
Add lysate, and bindHaloTag®^ fusion protein to Magne™ HaloTag®^ Beads.
Wash.
Cleave protein bound to bead using HaloTEV Protease.
Elute purified protein.
HaloTag®^ protein
Fused protein (^) HaloTEV Protease
Description and Principle
The Magne™^ HaloTag®^ Beads provide a convenient method to covalently capture HaloTag®^ fusion proteins with magnetic particles for protein pull-downs and puri- fication. HaloTag ®^ fusion proteins may be expressed in cell-based and cell-free systems. Cell lysates are mixed with Magne ™^ HaloTag®^ Beads and HaloTag® fusion proteins bound covalently to the beads. These magnetic beads offer a high-binding capacity (≥20mg/ ml) for purifying HaloTag®^ fusion proteins with low nonspecific protein binding. After washing, the protein of interest can be released from the beads by TEV Protease cleavage (either HaloTEV or ProTEV) at the optimized TEV recognition site. Proteolytic release yields the protein of interest, while the HaloTag®^ protein and HaloTEV Protease remain covalently attached to the beads (Figure 4.9).
Features and Benefits
- Simple: No centrifugation or vacuum is required once the cells are lysed.
- High Recovery: Binding capacity ≥20mg of purified HaloTag®^ fusion protein per ml of settled particles.
References
Verger A. et.al. (2013) The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members. Nucl. Acids Res. 41 (9), 4847–59. Yoshida S. et al. (2013) Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling. Circulation 2 (128), 60–71. Nagaki K. et al. (2012) Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco. Plant Cell Rep. 31 (4), 771–9.
Magnetic Affi nity-based Protein Purifi cation
4
Maxwell
® 16 Polyhistidine Protein
Purifi cation Kit
Automated purification of polyhistidine-tagged proteins from bacterial cultures and
mammalian and insect proteins.
Description and Principle
The Maxwell®^ 16 Polyhistidine Protein Purification Kit consists of cartridges prefilled with MagneHis™^ Ni-Particles and buffers that are used with the Maxwell®^ 16 Instrument to provide an easy method for efficient, automated purification of polyhisti- dine-tagged protein from various cells (Table 4.2). The Maxwell®^ Instrument is supplied with a preprogrammed purification procedure and reagent cartridges specifically designed to maximize simplicity and convenience. The instrument can process up to 16 samples in approximately 40 minutes.
Ordering Information Maxwell®^ 16 Polyhistidine Protein Purification Kit (Cat.# AS1060)
Features and Benefits
- Save Hands-On Time: Prefilled cartridges eliminate reagent preparation, multiple pipetting steps, centrifu- gation and additional sample manipulation.
- Consistent Results: Across all samples.
5933MA
Label side
Ridge side
Lysis Buffer
Wash Buffer Wash Buffer Wash Buffer Wash Buffer Empty
Sample
Plunger
Contents (^) User Adds:
MagneHis™ Ni-Particles
Figure 4.12. Maxwell 16®^ Polyhistidine Protein Purification Sample Cartridge
Figure 4.11. The Maxwell®^16 Instrument uses paramagnetic-particle technology to extract DNA, RNA or protein from up to 16 samples in less than 45 minutes. The paramagnetic particles and reagents are supplied in prefilled cartridges for faster separa- tions with less hands-on time.
Magnetic Affi nity-based Protein Purifi cation
Table 4.2. Sample Types and Maximum Processing Capacity per Cartridge.
Sample Type Processing Capacity Bacterial Culture Up to 20 O.D. 600 Mammalian Cell Culture Cells Up to 5 x 10
(^6) cells
Insect Cell Culture Cells Up to 5 x 10^6 cells Mammalian or Insect Cell Culture Medium 1ml
4
Magnetic Separation Devices
Manual or automated protein purification using magnetic particles.
Description
Promega offers a wide range of magnetic devices for separations from 0.5ml microcentrifuge tubes to 15ml or 50ml conical tubes, to 96- and 384-well standard and deep-well plates. The magnetic separation device for plates is useful for both manual and automated liquid-handling.
MagneSphere®^ Technology Magnetic
Separation Stands
MagnaBot®^ Magnetic Separation Devices
2312TA07_8A^ 3993TA02_3A MagneSphere®^ Technology Magnetic Separation Stand (two-position). Up to two sample volumes (50μl –1.0ml). Left to Right: Cat.# Z5331, Z5332, Z5333.
MagnaBot®^ 96 Magnetic Separation Device for 96-well standard- or deep-well-plates (20μl– 1.0ml). Cat.# V8151.
2309TA07_8A MagneSphere®^ Technology Magnetic Separation Stand (twelve-position). Up to two sample volumes (50μl–1.0ml). Left to Right: Cat.# Z5341, Z5342, Z5343.
2311TA07_8A
3417TA05_1A
PolyATtract®^ System 1000 Magnetic Separation Stand. One Sample volume (1-50ml). Cat.# Z5410.
MagnaBot ®^ 384 Magnetic Separation Device. Cat.# V8241.
MagnaBot ®^ II Magnetic Separation Device for 96-well PCR plate. Cat.# V8351.
Magnetic Separation Devices
