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Sample Concentration
- Add ~1.3 ml concentrating solution (Product # 66526) to a microfuge (Product # 69715)
- Add sample to a MINI unit and place membrane in contact with the concentrating solution in the microtube; cap the microtube; use at least 3:1 ratio of concentrating solution to sample.
- The sample will concentrate at 35-45 µl/hour
 MINI Unit in microtube for concentration.
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Dialysis Time Small Compound
We evaluated the dialysis rate per time of several other compounds in 3.5K, 7K and 10K Slide-A-Lyzer MINI Dialysis Units. A starting volume of 100 µl of 2 mg/ml ATP (Sigma, M.W. 551), 1 mg/ml Sulfo-NHS-Biotin (M.W. 556), 5 mg/ml bacitracin (Sigma, M.W. 1.4 kD) or 0.5 mg/ml vitamin B12 was dialyzed against 1 liter of water or TBS, pH 7.2. Samples of 5-10 µl were withdrawn from the Slide-A-Lyzer MINI Dialysis Unit at various time intervals to evaluate the dialysis process. Overnight dialysis samples remained undisturbed. ATP was monitored by A240 (Figure 5). Vitamin B12 was measured by A360 (Figure 6). Bacitracin was measured at A562 following BCA Assay (Figure 7). Biotin was determined using Wallac's Victor Microplate Reader and 2-anilinonaphthalene-6- sulfonic acid (Molecular Probes) assay. After overnight dialysis, ~1.3%, 0.8% and 0.2% biotin remained for the 3.5K, 7K and 10K Slide-A-Lyzer MINI Dialysis Units, respectively.
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Figure 5. Dialysis time course for 100 µl of 2 mg/ml ATP.
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Figure 6. Dialysis time course for 100 µl of vitamin B12.
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Figure 7. Dialysis time course for 100 µl of bacitracin.
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Scavenger Dialysis using the Slide-A-Lyzer MINI Unit
Scavenger dialysis can be used to selectively remove unwanted components of smaller molecular weight from a protein sample. The scavenger system has the following advantages:
- The protein never contacts the scavenger matrix
- No buffer changes are required~less hands-on time
- Less dialysate buffer is required
- Dialysis rates are improved by uptake of the unwanted component
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Scavenger Dialysis:
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Resin/Media Selection Guide:
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| Method |
Resin/Media |
Mobile Phase |
Sample Characteristic |
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Reverse Phase
(Partitioning)
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C18, C8, C2, cyclohexyl
(longer chain alkanes)
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Aqueous/Organic
Note: addition of 0.1% TFA opens most proteins (peptides and amino acids) exposing hydrophobic regions allowing binding to the RP adsorbent.
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Non-polar samples are retained and eluted with non-polar solvents
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Normal Phase
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Silica, Diol, NH2, CN
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Mix of Organic Solvents
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Polar samples are retained then eluted with polar solvents.
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Hydrophobic
Adsorbtion
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Similar to reverse phase, much less hydrophobic
(charcoal)
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Salt Solution
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Proteins
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Ion-Pair
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C8, C18, CN
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Aqueous organic buffer to control pH of ion-pair reagent
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Proteins
Samples associate with other compounds so their retention behavior is altered.
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Ion-Exchange
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Anion, Cation
(Silica, Alumina, Dowex, etc)
*see anion cation exchangers listed below
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Aqueous buffer with varied pH and ionic strength
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Organic ions, proteins, nucleic acids
Binds samples of opposite charge
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Affinity
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Various (table X)
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Various
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Various
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Chiral
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Cyclodextran
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Separation of left and right hand compounds (enantiomers)
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Ion-Exchangers, Buffers and Uses
Anion Exchangers:
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DEAE
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Diethylaminoethyl-
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6-9.5
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TEAE
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Triethylaminoethyl-
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NA
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QAE
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Trimethylaminoethyl- or other quartenary amines
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NA
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P
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Ampholytic polymers
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Complete pH range
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Cation Exchangers:
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CM
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Carboxymethyl-
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3.5-5.0
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P
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Phospho-
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5.5-7.5
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SP
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Sulphopropyl-
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NA
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S
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Sulphoethyl-
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NA
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Ion-Exchange Buffers:
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Cation Exchange
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Acetate
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pH 5
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MES
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pH 6
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MOPS
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pH 7
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Histidine
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pH 6
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Anion Exchange
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Diethanolamine
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pH 9
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Tris
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pH 8
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Imidazole
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pH 7
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Histidine
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pH 6
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Ion-Exchanger Used for Purification/Removal:
| Alumina |
Silica |
Charcoal |
Dowex |
| Alkaloids |
Phospholipase A |
Dyes |
Inositol Phosphates |
| Antibiotics |
Nucleic acids |
GTP from P |
Phospholipase D |
| Oils |
| Drugs |
| Plant extraction |
| Organic solvents |
| Gycosides |
| Hormones |
| Peroxides |
| Pyrogens |
| Steroids |
| Vitamins |
| Phosphates* |
*phosphate containing proteins, nucleotides, peptides
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Pierce Affinity Resins:
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Antibody fc region
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Protein A, G, A/G, T-gel™ Absorbent, Ni-IDA
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Antibody light chain (k)
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Protein L
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Carbohydrate
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Lectins
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Biotin
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Streptavidin, Avidin, NeutrAvidin™ Protein
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Histidine, (phosphate)
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Nickel Activated Imminodiacetic Acid, (Iron)
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GST fusion
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Glutathione
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Endotoxin Remover
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Detoxi-Gel™
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Detergent Removal
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Extracti-Gel Gel (OTG, CHAPS, SDS, Triton X, Tween 20, lysis buffers*)
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Scavenger Dialysis Application:
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Scavenger removal of fluorescent dye from fluorescent conjugated his-tag and his-region proteins using the Slide-A-Lyzer MINI Unit
Activated fluorescent reagent was covalently coupled to the 6his-tag of a fusion protein in A) lysate and B) purified form. The activated fluorescent reagent was also covalently conjugated to the C) his-region of an antibody. After conjugation, the fluorescent conjugates (100 µl of 0.1 mg/ml 6his-uricase or 0.5 mg/ml GtaRb antibody) were placed in 10K SAL MINI Units. The SAL MINIs were then placed in a microtube containing ~30 mg of acidic alumina and ~1.3 ml TBS. The MINI in the microtube was capped and left standing overnight at RT. All unconjugated dye was bound to the alumina resin at the bottom of the microtube. The conjugates were removed from the MINI, diluted then electrophoresed on a Novex 10% Bis-Tris (MES) gel. After electrophoresis, the gel was placed briefly in a clearing buffer and then imaged with CCD camera.
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Equilibrium Dialysis using the Slide-A-Lyzer MINI Unit
Equilibrium dialysis can be accomplished using the Slide-A-Lyzer MINI Unit and 48-well microplates. The Slide-A-Lyzer MINI holds a maximum volume of 0.5 ml. The well volume for the 48-well microplates are 0.3-0.6 ml (0.3-0.5 ml Costar #3548, & 0.4-0.5 ml Falcon #1178).
*Packard's Fusion™ Universal Microplate Analyzer has 48-well plate read capability.
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Books
Protein-Ligand Interactions A Practical Approach
Volume 1: Hydrodynamics and Calorimetry Edited by S.E. Harding and Babur Chowdhry
Chapter 2. Equilibrium dialysis and rate dialysis, Bent Honoré
Journal References
- Sarmiento, F., et al. (2000). The interaction between catalase and nafcilin by equilibrium dialysis measurements. Eur. Biophys. J. 29(4-5), 277.
- Plum, A., et al. (1999). Determination of in vitro plasma protein binding of insulin aspart and insulin detemir by equilibrium dialysis. Diabetologia 42(1) A236.
- Karim, S., et al. (1998). The effect of hyaluronan on the binding of diclofenac to human albumin using equilibrium dialysis. J. Pharm. Pharmacol. 50(1), 197.
- Kiehr, B., et al. (1998). Plasma protein binding: An iterative approach of performing equilibrium dialysis. Experimental and Toxicologic Pathology 50(2), 114-115.
- Ottiger, C. and Wunderli-Allenspach, H. (1997). Partition behaviour of acids and bases in a phosphatidylcholine liposome-buffer equilibrium dialysis system. European J. Pharm. Sci. 5(4), 223-231.
- Alexandre, H, et al. (1997). Interactions between toxic fatty acids for yeasts and colloids, cellulose and yeast ghost using the equilibrium dialysis method in a model wine. Food Biotechnol. 11(1), 89-99.
- Creutz, C. (1996). Equilibrium dialysis experiment: An interactive stimulation. American Journal of Botany 83(6), 214.
- Wilson, B. and Lindahl, P. (1999). Equilibrium dialysis study and mechanistic implications of coenzyme A binding to acetyl-CoA synthase/carbon monoxide dehydrogenase from Clostridium thermoaceticum. J. Biol. Inorg. Chem. 4(6), 742-8.
- Chattoraj, D. et al. (1999). Standard free energies of binding of solute to proteins in aqueous medium. Part 2. Analysis of data obtained from equilibrium dialysis and isopiestic experiments. Biophys. Chem. 77(1) 9-25.
- Chow, H., et al. (1998). A modified equilibrium dialysis technique for measuring plasma protein binding: experimental evaluation with diazepam and nortriptyline. Pharm. Res. 15(10), 1643-6.
- Sinha-Hikim, I., et al. (1998). The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women. J. Clin. Endocrinol. Metab. 83(4), 1312-8.
- Okabayashi, T., et al. (1996). Free thyroxine concentrations in serum measured by equilibrium dialysis in chronic renal failure. Clin. Chem. 42(10), 1616-20.
- Bizouarn, T., et al. (1996). The binding of nucleotides to domain I proteins of the proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli as measured by equilibrium dialysis. Eur. J. Biochem. 239(3), 737-41.
- Jaume, J. (1996). Extremely low doses of heparin release lipase activity into the plasma and can thereby cause artifactual elevations in the serum-free thyroxine concentration as measured by equilibrium dialysis. Thyroid 6(2), 79-83.
- Girard, I. and Ferry, S. (1996). Protein binding of methohexital. Study of parameters and modulating factors using the equilibrium dialysis technique. J. Pharmaceut. Biomed. Anal. 14(5), 583-91.
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