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MagListo™ His-tagged Protein Purification Kit (5 reactions)
MagListo™ His-tagged Protein Purification Kit (5 reactions)
MagListo™ His-tagged Protein Purification Kit allows easy purification of His-tagged proteins through magnetic separation by Ni-NTA Magnetic Nanobeads and buffers. With an average diameter of 400 nm, the beads have large surface areas with excellent binding capacity and can be recovered quickly without any loss through their strong magnetic force. These characteristics can quickly reduce the experimental time and the increase the yield. About 3~4 mg of 6x His-tagged proteins can be bound on to about 1 ml of Magnetic Nanobeads.
Cell-shredded samples can be prepared in advance and processed according to the manual of this kit to purify 6x His-tagged protein.
※ Note 1. The neodymium magnet included in this kit can be used for purification. 2. This kit can be purified using centrifuge without neodymium magnet. 3. Bioneer's MagListo™-2(TM-1010), magnetic particle purification system, can make it easier and faster to purify 8 samples at a time.
1. Protein purification using MagListo™ His-tagged Protein Purification Kit
2. Protein purification of various sizes from 25 kDa to 94 kDa using MagListo™ His-tagged Protein Purification Kit
Figure 2. Protein size effect on purification. - M : Protein broad marker (Bioneer, D-2010) - 1 : Size 94 kDa his-tagged protein - 2 : Size 67 kDa his-tagged protein - 3 : Size 38 kDa his-tagged protein - 4 : Size 27 kDa his-tagged protein - 5 : Size 25 kDa his-tagged protein
3. Comparison with other companies' magnetic bead products
Figure 3. Comparison with other products. - M : Protein broad marker (Bioneer, D-2010) - L : Loading whole sample - 1 : MagListo™ His-tagged Protein Purification Kit (K-7200) - 2 : Company A - 3 : Company B - 4 : Company C - 5 : Company D
Currently, the molecular weights (MW) of synthesizable proteins ranging from 10 kDa ~150 kDA were tested and validated. Though it is theoretically possible, it is not recommended to go over or under that scale.
Figure 1. SDS-PAGE data of various proteins synthesized from various templates M: AccuRapid™ Protein Size Marker (Broad) (Cat. No. D-2010, Bioneer) 1: CalmL3 (17.5 kDa) 2: DUSP3 (22 kDa) 3: CAT (24 kDa) 4: AcGFP (29 kDa) 5: ERFP (31 kDa) 6: EF-Ts (34 kDa) 7: VF (45 kDa) 8: BM3 (117 kDa)
The time it takes to obtain the proteins depends on the synthesized kit. For ExiProgen™ EC Protein synthesis Kit, it takes 6 hours for the expression to be completed. Refer to the table for more information on other kits. Additionally, after the final purification step, ExiProgen™ stores the target proteins in low temperature from 4°C to 8°C inside the reaction block. Even when the device is operated overnight, those can be also stably kept without separate refrigeration devices.
* It is recommended to use the Manual Kit for simpler controlling of expression volumes if only small scales of protein synthesis are required.
The template DNA should have the structure of T7 promoter, RBS, His-tag and T7 terminator as shown below. Depending on where the His-tag is attached, you can choose between two different types of expression, folding, and separation purification. At this time, the template type can be either plasmid DNA or PCR product. When preparing with a PCR product, it is advantageous to prepare several types of templates quickly, but the yield of protein synthesis may be slightly lower than that of plasmid DNA. Especially, when using SECF method for long time reaction for large-scale expression, the stability of template is important, so we recommend plasmid template.
1) When plasmid DNA is used as a template, It is recommended to use pBIVT vector (Cat. No. K-7350, Bioneer) which is exclusive to in vitro translation, but T7 system, RBS, and His-tag for purification can also be used. Using the AccuPrep® Nano-Plus Plasmid Mini Extraction Kit (Cat. No. K-3112, Bioneer), cloned plasmid DNA can be isolated with high-yield, high-purity only within 10 minutes
2) When using a PCR product as a template, ExiProgen™ ProXpress PCR Template Kit (Cat. No. K-7400, Bioneer) can be used for amplification of the target gene for preparing large quantities of the templates by PCR without the cloning step. For the best results, the purity of the acquired template DNA should be in the range of A260 / 280 ratio of 1.8 - 2.0.
Table. Related Products
Plasmid template generation by cloning
1) pBIVT Vector (Cat. No. K-7350)
2) pBT-7 vector (Gene synthesis service)
3) Other vector may also be used. (ex. pIVEX, pK7, pET vector series)
2) ExiProgen™ Protein Expression Optimization Kit (Cat. No. K-7410)
As the quantity of template DNA affects the efficiency of protein synthesis, it is very important to use an appropriate amount. Since the number of plasmid copies is important for the template DNA used, it should be quantitatively proportional to the size of DNA. In general, the amount of plasmid DNA added to 750 µl of expression volume is 1 µg/kb while the amount of linear DNA needed for the same expression volume is 500 ng (≤1 kb) or 1 µg (>1 kb). Please refer to the answer from Question 2) for more details.
If you want an optimal protein synthesis, we recommend an experiment that screens the amount of expressed protein according to the amount of template DNA used.
(Ex) An experiment using AccuRapid ™ Cell-Free Protein Expression Kit (Cat. No. K-7250) to optimize template DNA usage.
1. Prepare AccuRapid ™ Cell-Free Protein Expression Kit.
Expression volume: 45 µl
2. Prepare template DNA of the following concentrations (per 1 kb of plasmid DNA).
Standard DNA usage (1.34 µg/ml): 60 ng
DNA usage for tests: 120, 240, 360, 480, 600 ng
3. Proceed with the reaction referring to the manual of AccuRapid ™ Cell-Free Protein Expression Kit.
4. Verify the optimal amount of template DNA through SDS-PAGE analysis.
5. Get the optimal amount of DNA by converting from the expressed volume of the protein synthesis kit you are using (with the equation below).
Optimal DNA amount (ng) * expression volume of the kit you are using (µl) / 45 (µl)
Since the protein synthesis kit uses E. coli bacterial expression system, if the templates are optimized for E. coli codons, the protein expression levels and the success rates can be increased.
The purification methods used in ExiProgen™ utilize average diameter or 400 nm Silica Magnetic Nanobeads developed with Bioneer’s proprietary technology. On the surface of the beads, Ni-NTA is firmly immobilized, allowing purification of proteins by affinity purification methods through the strong binding of His-tag of the target proteins. These processes are fully automatic in ExiProgen™.
Due to the tertiary structure of the final product of proteins, if the His-tag at the N- or C-terminal is not exposed, to the surface, Ni-NTA can’t recognize it, lowering the purification efficiency. Thus, it is important to identify the protein structure and undergo a thorough check when designing the template DNA. If the protein structure is not known, it is advised test the expression and purification efficiency both templates with His-tag attached on N- or C- terminal. If those cannot be performed at both ends, it is recommended to increase the number of His-tag histidine to enhance them. If this method also does not work, then try attaching another protein which helps the protein expression to the N-terminal to produce a soluble fusion protein.
We are continuing to develop an efficient expression system, so please contact us if the protein expression fails.
Most of the time, this is caused by poor protein expression. It is recommended to use a manual kit AccuRapid™ to find the optimal protein expression condition on a small scale. Try running the same condition in ExiProgen™ for improved efficiency. If the expression level is high but the purification level is low, try changing the position of His-tag as explained in Question 7.
Each ExiProgen™ protein synthesis kit is consisted of two boxes: Kit ① and Kit ②.
It is highly recommended to store the purification kit (Kit ①) at 4℃ ~ 8℃ and Expression Kit (Kit ②) at -20℃ ~ -70℃. For the long-term storage, it is adviced to keep the E. coli extract included the Expression Kit (Kit ②) at -70℃.
For more details, please refer to the box and the user manual.
The cartridges is designed to be used up to 4 times. However, it is not recommended to use them overnight if you want to do so.
Store the them at the recommended temperature immediately after the ExiProgen™ operation. Conceal with cartridge covers and store cartridge ① at 4℃ to 8℃ and cartridge ② to -20 ° to -70 ° C.
The performance of the E. coli extract will be decreased on the occasion of repeated freezing and thawing. As it is packaged in a single tube for one-time use, please make sure to check the kit components and use it according to your experimental plans.
Expression of soluble protein is poor. Optimize the expression level of the soluble target protein by adjusting temperature of fermentation, induction time and concentration of inducer.
Protein forms inclusion body. If the protein forms inclusion bodies, it is unable to bind to the Ni-NTA magnetic beads. Optimize the expression process to minimize protein aggregation. Or purify aggregated protein under denaturing condition.
6x His-tag does not bind to the magnetic beads. If the structure of 6x His-tag is partially hidden, it is unable to bind to the Ni-NTA magnetic beads. Move the tag to the opposite end of protein. Or purify the protein under denaturing conditions. If the binding conditions are incorrect, optimize both pH value and imidazole concentration of the binding buffer. The binding buffer provided in the kit contains 10 mM of Imidazole, and its pH is 8.0. For a more detailed information, refer to the manual.
Target protein does not elute in elution process. Elution buffer conditions are too mild to elute protein. Increase imidazole concentration or decrease pH value. The elution buffer provided in the kit contains 500 mM of Imidazole, and its pH is 8.0. For a more detailed information, refer to the manual.
Buffer conditions are incorrect. Optimize pH value and increase imidazole concentration of binding/washing buffer to avoid unspecific interaction. The binding/washing buffer provided in the kit contains 10 mM of Imidazole, and its pH is 8.0. For a more detailed information, refer to the manual.
Insufficient washing step. Increase volume of washing buffer.
Column is too large. Reduce the amount of Ni-NTA resin to reduce unspecific interaction. The Ni-NTA magnetic nanobead solution provided in the kit contains 100 mg of beads per 1 ml.
Buffer conditions are incorrect. Lower the concentration of imidazole or increase the pH value slightly. The washing buffer provided in the kit contains 10 mM of Imidazole, and its pH is 8.0. For a more detailed information, refer to the manual.
6x His-tag weakly bind to the magnetic beads. If the structure of 6x His-tag is partially hidden, it might detach from Ni-NTA beads during the washing step. Reduce stringency of the washing buffer. Or purify under denaturing conditions. The washing buffer provided in the kit contains 10 mM of Imidazole. For a more detailed information, refer to the manual.