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Millipore Technical Publications



Product Catalogue
Protocol: Concentration, Desalting, and Buffer Exchange with Amicon Ultra or Microcon Centrifugal Filters
Catalogue Number: WWW-UF
Year:2005
Introduction
Amicon centrifugal devices from Millipore are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free from bound species.


Millipore centrifugal concentrators provide fast, convenient, high-recovery alternatives to dialysis and ethanol precipitation. Sample dilution, often associated with spin columns, is not a problem. Salt transfer across the membrane is efficient and independent of microsolute concentration or size.

Millipore’s Amicon Ultra-4 and -15 centrifugal filters are designed for high speed with high recovery. The devices incorporate low-binding Ultracel regenerated cellulose ultrafiltration membrane for sample concentration and purification of solutions containing dilute or purified protein solutes, antigens, antibodies, enzymes, or microorganisms. Their speed and excellent recovery make them ideal for desalting and buffer exchange applications. One of the most common applications for Amicon Ultra devices is concentration and desalting of column fractions during protein purification by various chromatography methods.

Two examples below demonstrate the use of Amicon Ultra devices for high protein and enzymatic activity recovery.

Method
  1. Select the device with the appropriate NMWL and volume for the application.
  2. Add the sample to the reservoir of the centrifugal device.
  3. If the sample is smaller than the maximum volume, it can be diluted up to the maximum volume before the first centrifugation step. This will help increase the salt removal.
  4. Centrifuge at the specified g-force for the recommended amount of time.
  5. Remove the initial filtrate from the filtrate tube and set aside.
  6. Add enough buffer or water to the device to bring the sample volume up to 4 or 15 mL.
  7. Centrifuge again.
  8. Set aside the filtrate.
  9. Recover the concentrated, de-salted sample.
NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed.
Results
The transfer of salts across a membrane filter is independent of sample concentration or size. There is no change in the composition of the buffer when desalting using ultrafiltration. For example, a solution containing 500 mM salt still contains that concentration after the initial centrifugation. Adding another volume of salt-free buffer or water to the retentate and centrifuging again will reduce the salt concentration. This process, known as diafiltration, can be repeated to achieve maximum salt removal.

Diafiltration can also be used when it is desirable to have the sample in a different buffer. The sample is concentrated and then repeatedly diluted with the desired buffer and concentrated again.

As the results show in Tables 1.1–1.4 , the efficient design of the Millipore devices allowed 90% of the salt to be removed during the first centrifugation step. Typically, only one subsequent centrifugation step was needed to increase the typical salt removal to 99% with >90% recovery of the sample.

Protein purification by chromatography usually involves the collection of multiple column fractions, with only some of those fractions containing the protein of interest. After the fractions are combined, a protein concentration step is often required for protein storage, or concentration with buffer exchange may be needed for downstream separations.

Table 1.1 Removal of sodium chloride and recovery of protein with Amicon Ultra-15 device
Cytochrome c 0.25 mg/mL
Cytochrome c 0.25 mg/mL
BSA 1 mg/mL
BSA 1 mg/mL
lgG 1 mg/mL
NMWL
5 kDa
10 kDa
30 kDa
50 kDa
100 kDa
Spin
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
1
94.5
97.2
97.3
97.9
96.0
98.2
98.9
97.1
99.9
97.7
2
92.6
99.9
95.6
99.9
94.4
99.9
92.4
99.9
97.1
99.5
Three Amicon Ultra-15 devices of each cut-off were tested with 15 mL of solute. 500 mM NaCl was added to each solution. Each spin was performed at 4,000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q (Millipore) system. OD readings were taken at 410 nm for Cytochrome c and 280 nm for BSA and lgG.


Table 1.2 Removal of sodium chloride and recovery of protein with Amicon Ultra-4 device
Cytochrome c 0.25 mg/mL
Cytochrome c 0.25 mg/mL
BSA 1 mg/mL
BSA 1 mg/mL
lgG 1 mg/mL
NMWL
5 kDa
10 kDa
30 kDa
50 kDa
100 kDa
Spin
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
% Protein Recovery
% NaCl
Removal
1
100.0
95.2
97.0
93.0
96.4
96.4
98.5
92.1
100.0
99.2
2
95.8
97.9
95.6
99.5
93.7
99.7
99.3
99.7
99.4
99.7
Three Amicon Ultra-15 devices of each cut-off were tested with 4 mL of solute. 500 mM NaCl was added to each solution. Each spin was performed at 4,000 x g for 10 minutes. After the first spin, the retentate was brought up to 4 mL with ultrapure water from a Milli-Q (Millipore) system. OD readings were taken at 480 nm for Cytochrome c and 280 nm for BSA and lgG.


Table 1.3 Removal of riboflavin and recovery of IgG with Microcon filter device with Ultracel-YM membrane
Spin Number
% Riboflavin Remaining
% lgG Recovered
1
9
95
2
2
93
3
<1
93
4
<1
92
500 μL of a 50:50 mixture of riboflavin and lgG were spun in a Microcon 3K NMWL device at 12,000 x g for 75 minutes at room temperature in 55 ° angle rotor. After the initial spin, the retentate was twice diluted with 500 μL of PBS and spun again. After each spin, concentration of riboflavin and lgG in the filtrate and retentate were monitored.
Concentration of Indoleamine 2,3-Dioxygenase
Courtesy of Eduardo Vottero, University of British Columbia

Indoleamine 2,3-dioxygenase (IDO; MW 48,000) is a heme-containing enzyme that is the first and rate-limiting enzyme in human tryptophan metabolism. IDO processes 98% of the total tryptophan available in the human body and is critical in suppression of immunoresponse by blocking T-lymphocyte proliferation locally [Swanson et al, Am J Respir Cell Mol Biol [manuscript in preparation] (2003); Sarkhosh et al, J Cell Biochem 90, 206 (2003); Mellor et al, J Immunol 171, (2003)].

Recombinant IDO was expressed in E. coli BL21 (DE3) cells utilizing the pET 28a (+) vector system. In this system, a hexahistidyl tag was fused to fullsize IDO at the N-terminus with a spacer sequence and a thrombin cleavage site. The protein was purified by conventional His-tag purification methods and eluted with imidazole. The histidine tag was removed by thrombin cleavage. Final purification was done by gel filtration chromatography G-75. Amicon Ultra-15 centrifugal devices were used to concentrate the IDO fractions from an initial concentration of ~0.5 mg/mL to a final concentration of 10 mg/mL. Samples were analyzed by SDS-PAGE using a 12.5% polyacrylamide gel (Figure 1.1). In addition, it was shown that no IDO activity loss was observed after concentration using an Amicon Ultra device.

Figure 1.5 (below) PKR concentration results
Spin 1
Spin 2
Spin 3
Spin 4
  Starting Volume (mL)
15
15
15
15
  Starting Concentration (mg/mL)
0.229
0.229
0.169
0.169
  Volume of retentate (μL)
590
490
203
225
  Total amount of
  PKR (mg)
      Before UF
      After UF
      Filtrate


3.4350
3.1005
0.0000


3.4350
3.1548
0.0000


2.5320
2.4081
0.0000


2.5320
2.3983
0.0000
  % Recovery
90.26
91.84
95.11
94.72


Figure 1.1 (left) SDS-PAGE of purified indoleamine 2,3-dioxygenase before and after concentration using Amicon Ultra-15 centrifugal devices.
Concentration of PKR and Buffer Exchange
Courtesy of Peter A. Lemaire and Dr. James Cole, University of Connecticut

  
Figure 1.2 Comparison of PKR autophosphorylation
with buffer exchange by ultrafiltration and without
buffer exchange.
  

Human protein kinase R (PKR) is one of the major proteins induced by interferon as part of the host defense against viral infection1–4. PKR is synthesized in a latent form and is activated by autophosphorylation induced upon binding dsRNA. Once phosphorylated, active PKR phosphorylates the eukaryotic translation initiation factor elF2a leading to a block in protein synthesis in virally infected cells. PKR has been implicated as a participant in various signal transduction pathways associated with cellular processes including transcription7–9, differentiation10, apoptosis11, splicing14 and transformation5,6. However, difficulties in purifying PKR in large amounts has limited rigorous biophysical characterization of the mechanisms of PKR activation.

A high-yield prokaryotic expression system has been developed for PKR, and PKR has been purified using three chromatography steps on Agarose-Heparin, Agarose-Poly (I), Poly (C) and Sephacryl S-200 gel filtration columns. After the last step, PKR-containing column fractions were pooled and concentrated using Amicon Ultra-15 30K NMWL devices. The concentration step was necessary for long-term protein storage. Table 1.5 shows the protein recovery results obtained after four concentrations. Over 90% recovery was obtained and no protein loss to the filtrate was observed

Amicon Ultra-15 30K NMWL devices were also used for exchanging buffer for PKR autophosphorylation (activation) assay. 200 µL of 6.301 mg/mL PKR in Protein Storage buffer (20 mM HEPES, 1 M NaCl, 10 mM b-mercaptoethanol, 0.1 mM EDTA, 10% glycerol, pH 7.5) was diluted to 15 mL with Phosphorylation Buffer (20 mM HEPES, 50 mM Kcl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, pH 7.5) and re-concentrated three times using Amicon Ultra devices at 3000 x g for 20 minutes at 4 °C. The filtrates from the three steps were pooled and the total amount of protein in all samples was determined by UV absorption A280.

Protein activity was tested by autophosphorylation assay. The protein in the storage buffer was supplemented with 5 mM MgCl2 and the samples were allowed to undergo autophosphorylation at 30 °C for 20 minutes in the presence of 3 mM ATP and 3 µCi [y32P] ATP. The activity was determined by autoradiography and quantified by liquid scintillation counting. As shown in Figure 1.2, the activity of PKR when no buffer exchange was only 6% of that when buffer exchange step was performed. Hence, the UF successfully exchanged the buffer while maintaining the activity of the protein.

References

1.Samuel CE. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 1991;183(1):1–11.
2.Hovanessian AG. The double stranded RNAactivated protein kinase induced by interferon: dsRNA-PK. J Interferon Res 1989;9(6):641– 7.
3.Lebleu B, et al. Interferon, double-stranded RNA, and protein phosphorylation. Proc Natl Acad Sci USA 1976;73(9):3107–11.
4.Samuel CE. Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc Natl Acad Sci USA 1979;76(2):600–4.
5.Koromilas AE, et al. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science 1992;257:1685–9.
6.Meurs E, et al. Tumor supressor function of interferon-induced double-stranded RNA activated protein kinase. Proc Natl Acad Sci USA 1993;90:232–6.
7.Wong AH, et al. Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and doublestranded RNA signaling pathways. EMBO J, 1997;16(6):1291–304.
8.Cuddihy AR, et al. Double-stranded-RNAactivated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol 1999;19(4):2475–84.
9.Demarchi F, Gutierrez MI, Giacca M. Human immunodeficiency virus type 1 Tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR. J Virol 1999; 73(8):7080–6.
10.Petryshyn R, et al. Effect of interferon on protein translation during growth stages of 3T3 cells. Arch Biochem Biophys 1996;326(2):290 –7.
11.Barber GN. Host defense, viruses and apoptosis. Cell Death Differ 2001;8(2):113–26.
12.Tan SL, Katze MG. The emerging role of the interferon-induced PKR protein kinase as a apoptotic effector: A new face of death? J Interferon Cytokine Res 1999;19:543–54.
13.Balachandran S, et al. Activation of the dsRNAdependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling. EMBO J, 1998;17(23):6888–902.
14.Osman F, et al. A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR. Genes Dev 1999;13(24):3280–93.

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