eLabNotebook - Promega's Automated MagneHis* Protein Purification System on the Biomek® FX

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Promega's Automated MagneHis* Protein Purification System on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

Materials to Be Supplied by the User:

• Tabletop centrifuge capable of 1,500 x g.

• Deep Well MagnaBot* 96 Magnetic Separation Device (Promega Cat.# V3031)

• 1/4 inch Foam Spacer (Cat.# Z3301)

• 2.2ml deep-well, square-well culture plates (Marsh Cat.# AB-0932)

• 1.2ml deep-well, square-well culture plates (Marsh Cat.# AB-0564)

• U-bottom multiwell plates (Promega Cat.# A9161)

For Biomek® FX:

• Pyramid-bottom reservoir plates (Innovative Microplate Cat.# S30014)

• Beckman Coulter Orbital Shaker ALP

For Tecan Workstations:

• Tecan Te-Shake Orbital shaker (3mm orbit)

Sample Preparation Before Automated Processing: If the bacterial cell pellets are frozen, thaw at room temperature for 10–15 minutes before use.

For cell pellets, dilute the supplied FastBreak* Cell Lysis Reagent to 1X concentration with water before use. For cell cultures, the supplied 10X FastBreak* Reagent is added directly to the cell cultures at a 1:10 ratio (i.e., 100µl 10X Fastbreak* Reagent to 900µl culture volume).

A. Initial Deck Layout for the Biomek® FX

This is an example of the MagneHis* Protein Purification System deck layout on a Biomek® FX.Your specific deck layout may be different depending on your Biomek® FX configuration.

(Click to Enlarge)
Figure 1. Biomek® FX initial deck configuration.

ALP Name	Equipment
Tip Loader	200µl ART* Biomek® FX Tips
P1	200µl ART* Biomek® FX Tips
P2	200µl ART* Biomek® FX Tips
P3	Empty: Tip box swap spot
WS1	Tip Wash Station (96 channel; not necessary for method)
P4	96-well, round-bottom plate containing 45µl of MagneHis* Resin/well (labeled “MagneHis”)
P5	Empty
P6	Empty 96-well round-bottom plate (labeled “Final Eluate”)
P7	Pyramid-bottom reservoir plate containing 25ml FastBreak* Cell Lysis Reagent (10X concentration for cultures; 1X concentration for cell pellets) + 25µl DNase I, if desired (labeled “Lysis”)
P8	Pyramid-bottom reservoir plate containing 50ml MagneHis* Binding/Wash Buffer (labeled “Wash”)
P9	Pyramid-bottom reservoir plate containing 25ml MagneHis* Elution Buffer (labeled “Elution”)
P10	Empty. Reserved for plate movement
P11	Empty Marsh 1.2ml 96-well, round-bottom plate (labeled “Working Plate”) on a Deep Well MagnaBot* 96 Magnetic Separation Device with 1/4 inch Spacer
P12	Empty Marsh 2.2ml deep-well square-well plate to collect flowthrough (labeled Flow-thru”)
Orbital 1	Marsh 2.2ml deep-well square-well culture plate containing 900µl cell culture in suspension or cell pellets (labeled “Cell Plate”)

B. Biomek® FX Specific Pre-Run Recommendations

The Biomek FX® automated platform allows users the flexibility to configure the robot's deck according to need. Because of this flexibility in deck configuration, it is likely that the deck used for writing a Biomek® FX method will differ from an end-user’s deck. Therefore, it will be generally necessary to map an imported method onto an end-user’s deck configuration. To do this, follow the instructions provided in the document Deck Mapping on the Biomek® FX (www.promega.com/automethods/beckman/biomekfx/default.asp).

C. Description of Automated MagneHis* Protein Purification System

This overview describes general liquid handling steps required for the automated MagneHis* Protein Purification System and can be adapted to a variety of automated liquid-handling robots. For additional information about adaptation to liquid handling robots other than those referenced above, please see Section VIII, General Guidelines for Adaptation to Alternative Robotic Platforms.

	1.	*MagneHis Particle Dispense.** MagneHis* Ni-Particles are mixed in the reservoir, then 30µl aliquots are dispensed into a 1.2ml deep-well plate.
	2.	Cell Pellet Resuspension/Lysis Cell pellets contained in the deep-well 96-square-well culture plate are resuspended with FastBreak* Cell Lysis Reagent by a combination of mixing and shaking for 5 minutes. If processing cell pellets, 200µl 1X FastBreak* Reagent is used. If processing cell cultures, 100µl 10X FastBreak* Reagent is used. Note: to overcome sticky or ‘slimy’ cell lysates add 1µl/well of resuspended DNase I.
	3.	*Bind Protein to MagneHis Ni-Particles.** The lysate is added to the MagneHis* Ni-Particles to initiate binding of His-tagged proteins. The lysate is mixed well with the particles by shaking for 1 minute. After shaking, the plate is placed onto the Deep Well MagnaBot* Device to capture the particles. The supernatant, containing unbound materials, can be saved for analysis or discarded to waste.
	4.	Washes. Three washes are performed with the MagneHis* Binding/ Wash Buffer to remove residual impurities from the sample. During each wash, 100–150µl of MagneHis* Binding/Wash Buffer is added to the MagneHis* Ni-Particles. The particles are mixed well by shaking for 1 minute. After shaking, the particles are captured on the Deep Well MagnaBot* Device, and the supernatant is discarded to waste. This process is repeated 2 more times.
	5.	Elution. The samples are eluted from the particles by adding 100µl of MagneHis* Elution Buffer and mixing well for 1 minute on the shaker. After shaking, the particles are captured on the Deep Well MagnaBot* Device, and the supernatant is saved in a collection plate for analysis. VIII. General Guidelines for Adaptation to Alternative Robotic Platforms The MagneHis* Ni-Particles settle over time. It is recommended to thoroughly mix the MagneHis* Ni-Particles on the automated platform prior to dispensing into samples. Resuspension of the MagneHis* Ni-Particles can be accomplished by thorough mixing or shaking.

* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.