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Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration

Allen T. Phillips¹, Mark W. Signs¹

¹Pennsylvania State University, University Park, Pennsylvania

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 4.4

DOI:

10.1002/0471140864.ps0404s38

Online Posting Date:

January, 2005

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Mark Signs

Abstract

This unit includes a variety of dialysis and ultrafiltration techniques that can be used for desalting, concentration, or buffer exchange. Standard dialysis by diffusion across cellulose tubing is described as a technique for desalting or buffer exchange. Ultrafiltration under pressure can be used either for concentrating protein or, where the sample volume is replenished with a desired buffer, for desalting/buffer exchange (i.e., diafiltration). A variation is ultrafiltration by tangential flow, which serves the same purposes as ultrafiltration under pressure but is better suited to handling large volumes of solution. Yet another variation is an ultrafiltration technique employing centrifugal microconcentrators, which allows concentration of very small volumes of solution.

Keywords: ultrafiltration; dialysis; tangential flow filtration; diafiltration; microconcentration

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Unit Introduction
Basic Protocol 1: Desalting and Buffer Exchange by Dialysis Through Regenerated Cellulose Tubing
Basic Protocol 2: Concentration or Dialysis by Ultrafiltration Through Asymmetric Membrane Disks Under Pressure
Alternate Protocol 1: Diafiltration or Concentration by Tangential-Flow Ultrafiltration
Alternate Protocol 2: Microscale Concentration and Desalting with Centrifugal Ultrafilters
Commentary
Literature Cited
Tables

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Materials

Basic Protocol 1: Desalting and Buffer Exchange by Dialysis Through Regenerated Cellulose Tubing

Materials

Dialysis tubing: regenerated cellulose, molecular weight cut-off (MWCO) 12,000 (e.g., Spectra/Por Type 4, Spectrum) for general protein use or MWCO 3500 (e.g., Spectra/Por Type 3) for retention of small proteins, stored at 4°C in a tightly closed plastic bag
50% (v/v) ethanol in large wash bottle
10 mM EDTA, pH 8.0
0.05 M NaHCO₃
Protein solution (clarify by centrifugation if necessary)
Appropriate dialysis buffer

Large wash bottle
Weighted and unweighted tubing closures (Spectrum)
Small plastic funnel
Glass marble or glass beads (optional): rinse thoroughly with dialysis buffer before use
Conductivity meter and probe (optional)

Basic Protocol 2: Concentration or Dialysis by Ultrafiltration Through Asymmetric Membrane Disks Under Pressure

Materials

Protein solution (clarify by centrifugation if necessary)
N₂ gas cylinder and two-stage pressure regulator capable of controlling delivery up to 100 psi
5% ethanol

Ultrafiltration membrane (YM10, Amicon; Ultracel PLGC, Millipore; or PM10, Amicon) with MWCO >10,000 and diameter to fit stirred cell
Ultrafiltration unit, stirred-cell type (Amicon Series 8000, Millipore) with:
- Glass funnel with stem diameter to fit through cell fill hole (optional; used in certain cell models)
- Ultrafiltrate collection container (Griffin beaker with graduations)
- Tubing for connection to N₂ source
- Pressurized reservoir (optional; for use when sample size is large and protein is dilute, or when diafiltration is performed with constant readdition of new buffer; e.g., RG5 Fiberglass Reservoir, Amicon)

NOTE: Handle the membrane only with powder-free gloves and hold by the edge to avoid scratching the smooth, thin “skin” surface.

Alternate Protocol 1: Diafiltration or Concentration by Tangential-Flow Ultrafiltration

Materials

Dialysis buffer
Protein sample
0.2% (w/v) Terg-A-Zyme detergent (e.g., VWR), 40°C
0.05% (w/v) sodium azide

Pellicon XL 50 (Millipore) equipped with a membrane filter plate of cellulose (PLCGC, MWCO 10,000 or PLCTK, MWCO 30,000) or polyethersulfone (Biomax 30, MWCO 30,000)
Luer-to-barb connectors
4-mm-i.d. tubing (Tygon or silicone)
Sample and retentate collection reservoirs: containers capable of holding entire sample
Hose clamps, adjustable with screws
Permeate collection vessel: Erlenmeyer flask larger than the sample volume
Variable speed peristaltic pump equipped with pump head of 480 ml/min rated capacity (equivalent to that supplied with the Millipore Labscale TFF system)
10-ml syringe

Alternate Protocol 2: Microscale Concentration and Desalting with Centrifugal Ultrafilters

Materials

Centrifuge microconcentrator units: e.g., Microcon-10, -30, or -50 (MWCO 10,000, 30,000, and 50,000 respectively; Amicon); or Ultrafree-MC with MWCO 5,000, 10,000, or 30,000 (Millipore)
Microcentrifuge, 4°C, with either a fixed-angle or swinging-bucket rotor, capable of providing forces up to 13,000 × g

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Literature Cited

Literature Cited
	McPhie, P. 1971. Dialysis. Methods Enzymol. 22:23-32.
	Saltonstall, C.W. 1992a. Separations with dialysis and ultrafiltration: Theoretical and practical considerations. Am. Biotechnol. Lab. 10:32-34.
	Saltonstall, C.W. 1992b. Dialysis and ultrafiltration: Estimating quantitative separations. Am. Biotechnol. Lab. 10:18-20.
	Suelter, C.H. and Deluca, M. 1983. How to prevent losses of protein by adsorption to glass and plastic. Anal. Biochem. 135:112-119.
Key References
	Pohl, T. 1990. Concentration of proteins and removal of solutes. Methods Enzymol. 182:68-83.
A thorough review of the topics covered here.
	Scopes, R.K. 1994. Protein Purification: Principles and Practice, 3rd ed. pp. 14-21. Springer-Verlag, New York.
Covers concentration and dialysis in the context of protein purification techniques.
	Schratter, P. 1996. Purification and concentration by ultrafiltration. Methods Mol. Biol. 59:115-134.
Additional description of protein concentration methodology.
	Doonan, S. 1996. Concentration of extracts. Methods Mol. Biol 59:95-102.
Additional description of protein concentration methodology.
Internet Resources
	http://www.millipore.com/publications.nsf/dda0cb48c91c0fb6852567430063b5d6/21faf0f81edcbb68852568f00053d6a2/$FILE/ATTDMXFA/PC1001EN00.pdf
Millipore Protocol Note, No. PC1001EN00, regarding results from passivation of Microcon concentrators following treatment with a variety of agents.