Purification on an Acrodisc® Unit with Mustang® S Membrane 4.1.3

Protocol for Purification on an Acrodisc® Unit with Mustang S Membrane

A. Materials Required

1. Syringes (5-25 mL) with luer lock fittings.
2. Chromatography fittings to transition 1/8 inch tubing connections to male luer-lock inlet, and female slip luer fitting on the outlet (UpChurch or equivalent).
3. Degassed and filtered suitable buffers; Pump A, 10 mM MES-NaOH pH 5.5; and Pump B, 1 M NaCl in buffer A or 25 mM Tris HCl pH 8.0.

B. Ion Exchange Purification Can Be Carried Out by Two Approaches

1. Changing the pH of the buffer.
2. Introducing a counter ion into the loading buffer in the form of a salt gradient.
3. In both cases, proteins elute from the ion exchange surface because:
   1.  They become neutral or acquire the same charge as the ion exchange support.
   2. They are displaced by the presence of a small counter ion in the form of salt.
4. The two approaches are useful in developing an optimal purification strategy and are summarized in Table 4.5. Either strategy or a combination of both can be applied to the purification of components from a complex sample in the Acrodisc device format. These devices can be operated by syringe (step gradient elution) or pumped flow (stepped and gradient elution) using a chromatography workstation.

Table 4.5

Summary of Purification Options for Acrodisc Unit with Mustang S Membrane

 

C. Syringe Protocol

1. Before filling the syringe with sample, draw approximately 1 mL of air into the syringe. This will allow the air to follow the sample out of the syringe. This “air purge” minimizes fluid retention within the cartridge.
2. Fill the syringe with equilibration buffer A.
   Tip: Use of syringes smaller than 10 mL can generate excessive pressure on the cartridge, which may exceed maximum operating pressure.
3. Holding the filter device in one hand and the filled syringe in the other, secure (without excessive force) the filled syringe to the filter device with a twisting motion.
4. Apply gentle pressure to begin passing fluid through the device. (A gentle pressure helps assure maximum throughput.)
5. Collect the column effluent in 0.5 mL fractions. Measure the A280 to locate the protein peak.
   Tip: Protein rapidly elutes from the device and should be found in the first three fractions. Some slight dilution of the sample will occur during elution. If necessary, the sample can be concentrated in a centrifugal UF spin filter, such as a Nanosep® centrifugal device, with a 10K MWCO UF membrane.
6. Retained fractions can then be eluted by step gradient of buffer pH, up to 1.0 M salt, or a combination of both.
7. Each step of the gradient should be at least 2-5 column volumes (CV). Fractions of 0.5 mL should be collected. After protein has eluted, the device can be regenerated by 5 CV of 1.0 M NaCl followed by equilibration back to initial buffer conditions. 

D. Syringe Protocol on Chromatography Workstation

1. Place luer fitting adaptors onto the Acrodisc® device. Connect to a chromatography workstation.
2. Set flow to 100% buffer A at 1 mL/min and fill the device with fluid in the reverse flow direction to displace air from the device.
3. Reverse the flow and equilibrate the device for 5-10 CV of buffer A.
4. Load the sample up to a 2 mL volume onto the column at 1 mL/min flow rate. Monitor effluent at 280 nm.
5. Collect the column effluent in 1 mL fractions. Measure the A280 to locate the protein peak.
   Tip: Protein rapidly elutes from the device and should be found in the first three fractions. Some slight dilution of the sample will occur during elution. If necessary, the sample can be concentrated in a centrifugal UF spin filter, such as a Nanosep centrifugal device, with a 10K MWCO UF membrane.
6. Retained fractions can then be eluted by linear gradient of buffer pH, linear gradient up to 1.0 M salt, or a combination of both.
7. The volume of the gradient should be at least 5-10 CV. Fractions of 1 mL should be collected. After protein has eluted, the cartridge can be regenerated by 5 CV of 1.0 M NaCl followed by equilibration back to initial buffer conditions.

Application Data for Purification on an Acrodisc Unit with Mustang S Membrane

Resolution of a mixture of Cytochrome C and lysozyme at pH 5.5 is summarized in Figure 4.3. The data clearly shows a rapid (< 10 minutes) separation of the two proteins as well as resolved symmetrical peaks at a very high flow rate of 13 CV/min. The dynamic binding capacity for this Mustang S device is summarized in Figure 4.4. The cartridge capacity was calculated to be 8 mg lysozyme with a membrane media capacity of 52 mg/mL, which is very comparable to conventional particle-based media. The Mustang S Acrodisc device offers a very high flow rate. It is an equivalent capacity device which can yield high resolution separations. 

Figure 4.3

Acrodisc® Unit with Mustang S Membrane: Resolution of Cytochrome C and Lysozyme

The conditions used to generate data for the resolution graph above include buffer: 10 mM MES-NaOH pH 5.5; salt: 1 M NaCl in 10 mM MES-NaOH pH 5.5; gradient: 0 to 0.5 M NaCl in 50 CV; flow rate: 2.3 mL/min (13 cv/min); sample loading: 4% of total binding capacity.

Figure 4.4

Acrodisc Unit with Mustang S Membrane: Dynamic Binding with Lysozyme

A solution of 0.512 mg/mL lysozyme was pumped through the Acrodisc unit at 2.3 mL/min. Breakthrough occurred at 8.0 minutes and was calculated as 52 mg/mL using: flow rate (2.3 mL/min) X initial protein concentration (0.524 mg/mL) X time (8.1 minutes) membrane bed volume of Mustang S membrane in 25 mm Acrodisc unit (0.18 mL).

Ordering Information for Purification on an Acrodisc® Unit with Mustang S Membrane

Acrodisc Unit with Mustang S Membrane

Part Number	Description	Pkg
MSTG25S6	0.8 μm, 25 mm, non-sterile, blister packs	10/pkg

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