Bio-rad 732-4620, 732-4624, 732-4622 User Manual

Bio-Scale™Mini Profinity™GST Cartridges, 1 and 5 ml

Instruction Manual

Catalog # 732-4620 732-4622 732-4624

Table of Contents

Section 1...Introduction ........................................1

Section 2 Product Information............................2

Section 3 Connection to Low-Pressure

Chromatography Systems..................6

Section 4 Connection to Medium- and High-

Pressure Chromatography Systems .10

Section 5...Buffers and Methods........................12

Section 6 Quick Solubility Screening

Protocols..........................................13

Section 7 Preparation of E. coli Lysates ...........17

Section 8 Preparing a Cartridge, and

Subsequent Purification....................19

Section 9 Scaling Up........................................23

Section 10 Regenerating, Cleaning,

Sanitizing, and Storage.....................24

Section 11 Troubleshooting Guide......................26

Section 12 Ordering Information.........................29

Section 13 References .......................................31

Section 14 Legal Notices ...................................32

Section 1 Introduction

Bio-Scale Mini GST cartridges are convenient, disposable, prepacked low-pressure chromatographic cartridges. Bio-Scale Mini cartridges offer both increased run-to-run reproducibility and high purity of protein through a patent-pending column design and novel resin technology. Compatible with aqueous buffers most commonly used for protein purification, Bio-Scale Mini cartridges offer improved performance for protein separation needs.

Profinity glutathione support is based on Bio-Rad's proprietary UNOsphere

™

technology (US patent 6,423,666) for capture and purification of glutathione S-transferase (GST)-tagged proteins. Its ligand density has been optimized for maximum capture of target proteins. Ideal for scale-up, the Profinity glutathione support’s open pore structure is ideal for purifying proteins of a wide molecular weight range. (Smith DB et al., 1988)

Bio-Scale Mini GST cartridges are packed with

Bio-Rad's innovative Profinity GST resin. Structural characteristics such as the polymeric nature, optimized ligand density, and open pore structure of the Profinity GST bead result in superb mechanical strength and performance of the prepacked cartridges.

Section 2 Product Information

Bio-Scale Mini cartridges are disposable, easy-to-use, prepacked chromatographic cartridges supplied ready for use in convenient 1 ml and 5 ml sizes. Cartridges are available for a variety of chromatographic techniques, including desalting, ion exchange (IEX), and affinity (AC) chromatography. See Ordering Information for a listing of the complete Bio-Scale Mini cartridge product line.

Bio-Scale Mini cartridges are quickly connected to liquid chromatography systems or luer-end syringes. The cartridges can be used with any liquid chromatography system capable of setting a high pressure limit of 45 psi (equivalent to 3 bar or

300 kPa). Alternatively, luer fittings offer convenient connection directly to a Luer-Lok syringe for quick, one-step purification.

Table 1 Bio-Scale Mini GST cartridge specifications

Sizes 1 ml and 5 ml bed volumes

Dimensions 1 ml: 40 mm length x 5.6 mm inner

diameter

5 ml: 40 mm length x 12.6 mm inner diameter

Maximum pressure tolerance 45 psi

Recommended flow rates 1 ml: 1.0–2.0 ml/min (240–480 cm/hr)

5 ml: 5–10 ml/min (240–480 cm/hr)

Maximum flow rate 1 ml: 6 ml/min (1,440 cm/hr)

5 ml: 20 ml/min (963 cm/hr)

Fittings Female luer inlet and

male luer outlet

Column material Polypropylene

Frit material Polyethylene (HDPE)

Shipping conditions 20% ethanol

Storage recommendations 20% ethanol or 2% Benzyl Alcohol

Autoclavability Not autoclavable

Table 2. Profinity GST Resin Specifications

Functional ligand Glutathione Derivative

Base bead UNOsphere

Particle size range 45–90 µm

Mean particle size 70 µm

Functional group density ≥60 µmol/g

Dynamic binding capacity* >11 mg/ml

Recommended linear flow rate <600 cm/hr at 25°C

Maximum operating pressure ≥43 psi

Chemical compatibility See table 3

Storage 4–8°C

Shelf life in 20% EtOH >1 year at 4–8°C

Operational temperature 4–40°C

* 60% Breakthrough for a purified 51 kD GST fusion protein at 0.5 ml/min.

Note: Dynamic binding capacity is a function of a number of factors including pH, flow rate, and sample temperature.

Profinity GST cartridges are compatible with aqueous buffers most commonly used with GST purification techniques.

Table 3. Buffer and Chemical Compatibilities for Profinity GST cartridges

Reagent Stability

Buffer reagents

Tris 50 mM HEPES 50 mM MOPS 50 mM Sodium or potassium phosphate 50 mM

Chelating agents

EDTA, EGTA 5 mM

Sulfhydryl reagents

β−Mercaptoethanol 30 mM DTT 5 mM TCEP 10 mM

Detergents

Nonionic detergents (Triton, Tween, NP-40) 5% Cationic detergents (CTAB) 1% Zwitterionic detergents (CHAPS, CHAPSO) 5% Anionic detergents (SDS, Sarkosyl) 1%

Denaturing agents (for cleaning only)

Guanidine-HCl 6 M Urea 8 M

Other additives

NaCl 2 M MgCl

100 mM

CaCl

10 mM Glycerol 20% Ethanol 20% Citrate 80 mM

Section 3 Connection to Low-Pressure Chromatography Systems

Bio-Scale Mini cartridges are ideal for use with Bio-Rad's BioLogic™LP chromatography system, Econo™Gradient pump, the patented* Model EP-1 Econo pump, and all low-pressure chromatography instruments. Bio-Scale Mini cartridges can be conveniently connected directly to the system using the luer fittings on the cartridge.

1. Install 1.6 mm inner diameter (ID) tubing in the pumphead. Adjust the platen pressure screw (on pumphead) using a screwdriver or coin. Turn the screw counterclockwise as far as it will go, then turn clockwise three full turns. Assemble with fittings and lock rings as shown in Figure 1.

* US patent 5,135,658

(Use orange lock rings and medium size barb fittings with 1.6 mm tubing.)

Fig. 1. BioLogic LP system setup.

2. To maximize gradient accuracy and to apply samples efficiently, install 1.6 mm ID tubing from the pump to the MV-6 sample inject valve (if available). If using the MV-6 sample inject valve, turn the knob counterclockwise as far as it will go so it will now correspond to the printed diagram on the valve (see Figure 2).

Platen pressure

screw

Lock-ring

Tubing

Luer fittimg

See detail

456

C .

Alarm

Fig. 2. Connecting to an MV-6 valve.

3. Connect the inlet of the cartridge to the male luer fitting on the MV-6 sample inject valve (see Figure 2). If not using the MV-6 sample inject valve, connect a barb to male luer fitting on the

1.6 mm ID tubing, then connect to the top of the female luer on the Bio-Scale Mini cartridge. For optimum performance, a cartridge should be mounted vertically with the arrow on the cartridge pointing downward (see Figure 3).

BIOLOGIC LP

SYSTEM OR ECONO

GRADIENT PUMP

SAMPLE LOOP

INJECT PORT

MV-6

INJECT

VALVE

FILL

COLUMN

WASTE