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Automation Method for Protein Purification by using Promega's MagneHis* Protein Purification System
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Hiromasa Kashimori, Beckman Coulter K.K., Japan

Abstract

Poly-histidine tag is one of the most popular affinity tags used for the purification of expressed recombinant proteins. Its popularity as a purification method is based on the fact that it is relatively simple to incorporate and faster than other affinity tags. We demonstrate here the ease of automation and results of the automated high-throughput protein purification system using Beckman Coulter's BiomekŪ 2000 and Promega Corporation's MagneHis Protein Purification System.

System and Devices

The base automation platform for the His-tag purification system is Biomek 2000 Laboratory Automation Workstation (Figure 1) with a DPC MicroMix* 5 orbital shaker on the right side of Biomek 2000 instead of the left-side module (Figure 2). Promega's MagnaBot* magnetic separation device is integrated on the deck of the Biomek 2000 for affinity capture of the his-tagged proteins via MagneHis magnet bead separation in liquid phase. The MP200 tool is used for liquid pipetting and Gripper tool is used to transport the plates on the deck.


Figure 1. Deck of the Biomek 2000 Workstation set for automation of the MagneHis protein purification system.


Figure 2. DPC MicroMix 5 orbital shaker on the right side of the deck of the Biomek 2000.

Protocol

  1. Add Lysis buffer to harvested E.Coli cell suspension.
  2. Add MagneHis magnetic beads to each well and mix.
  3. Pull the magnetic beads out of solution using the MagnaBot.
  4. Remove supernatant by aspiration.
  5. Wash the magnetic beads three times using the binding/wash buffer.
  6. Finally, elute the His-tagged proteins from the MagneHis magnetic bead with addition of the elution buffer.

Results and Conclusions:

The total run time for the processing of 96 samples in a single 96-well plate is 1.5 hours. As is shown in Figure 3, there are no additional protein bands observed in the SDS-PAGE gel after purification. We conclude from this data that the method for the automation of the MagneHis kit provides sufficient purification of expressed protein for structural and functional analysis. While this analysis was done at ambient temperature, it is also possible to integrate a cooling device on the deck of the Biomek 2000 use the purification of an expressed temperature-sensitive protein.


Figure 3. Resolution of His-tagged proteins after purification using the MagneHis purification system.

Figure 3 Legend:

M = Molecular Weight Ladder; A = sample from well A5; B = sample from well B5; C = sample from well C5; D = sample from well D5; E = sample from well E5; F = sample from well F5; G = sample from well G5; H = sample from well H5; I = sample from well A6; J = sample from well B6; K = sample from well C6; L = sample from well D6; M = sample from well E6; N = sample from well F6; O = sample from well G6; P = sample from well H6.

* All trademarks are the property of their respective owners.

For Research Use Only; not for use in diagnostic procedures.

For comments or questions about T3 Update, please contact David Daniels, Ph.D., editor.

 
 
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