Filter Centricon

Sample preparation 500 xL Milk + 500 p,L MeCN MeOH water 40 20 40, vortex for 10-15 s, filter (Centricon-10, molecular mass cut-off filter 10000 daltons) with centrifuging at 2677 g for 30 min, iiyect a 10-100 p,L aliquot of the ultrafiltrate. 

Figure 12.5 A section of the proton NMR spectra of a biopharmaceutical protein product, spiked with 10 pg/ml of acetate and MES, before (upper spectrum) and after (bottom spectrum) the protein was removed by filtering the sample with Centricon-10. The acetate and MES are recovered after the filtration to remove the protein.

Step II Diafiltration. Extracts to be fractionated by HPLC were diafiltered into buffer A using Centricon 10 microconcentrators (Amicon Div., Grace Co., Danvers, MA) and then passed through 0.22 jim filters. 

Centricon YM-3 centrifugal filter devices (Millipore, Bedford, MA). 

Notes. A Centricon-30 tube may be substituted for use with very small DNAs that may not be retained by the Centricon-100 tube. Millipore (Bedford, MA) MC 100,000 MWL filter units (UFC3 THK 00) may be substituted if desired. Because of their smaller volume, additional dilution steps may be required to desalt the ssDNA before sequencing. For brands of streptavidin magnetic beads whose binding efficiency for biotinylated DNA may decrease as the length of the DNA increases, it may be essential to reduce the primer concentration via this prespin step. 

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

The protein solution is concentrated and desalted against KP 6.2 in Centricon-10 filter units (Amicon, Danvers, MA) as directed by the manufacturer. 

Sample preparation Senim. 500 p.L Serum + 500 p,L MeCN 100 mM NaOH 50 50, vortex for 10-15 s, filter (Amicon Centricon-10, 10000 Daltons) while centrifuging at 2677 g for 30 min, iiyect a 30-120 pL aliquot of the ultrafiltrate. Tissue. Cut up prostate tissue with a scalpel. Weigh out 100-130 mg tissue, make up to 500 pL with MeCN 100 mM NaOH 50 50, sonicate for 30 min, filter (Amicon Centricon-10, 10000 Daltons) while centrifuging at 2677 g for 30 min, iiyect a 80-120 pL aliquot of the ultrafiltrate. 

Centrifugation With the concentration devices from the Amicon company, the solution to be concentrated is centrifuged in a fixed-angle rotor over a filter (max. 5,000 g). The concentrate collects above the filter and can be transferred into an Eppendorf tube in a second centrifugation step. The different systems (Microcon, Centricon, Centriprep) concentrate 0.5 to 15 ml of source solution into 5 to 500 pi. Depending on the filter, the process takes between 10 minutes and 3 h. The company offers filters with MG limits from 3 to 100 kd. 

Sample preparation Prepare a column as follows. Swirl Chelating Sepharose Fast Flow resin (Pharmacia) in its bottle, add it to a polypropylene column to give a bed volume of 1.0-1.2 mL, wash 3 times with 2 mL portions of water, wash with 2 mL 10 mM copper sulfate, wash with two 2 mL portions of water. Centrifiige 5 mL milk at 10° at 1500 g for 15 min, remove the lower layer and add it to 10 mL succinate buffer, mix, centrifuge at 1500 g for 30 min, add the supernatant to the column. Wash with 2 mL succinate buffer, wash with 2 mL water, wash with 2 mL MeOH, wash with 2 mL water, wash with 700 pL citrate/phosphate buffer (be careful not to disturb bed), elute with 2.5 mL cit-rate/phosphate buffer (column is white and eluate is blue). Filter (Amicon Centricon 30,... 

Measurement and Assignments of the Raman Bands that Aid the Modeling of A-Track DNA Deoxyoligonucleotides and polymers containing only adenine (A) and thymine (T) were purchased from Pharmacia and were desalted using Centricon-3 filters (Amicon) and then concentrated to dryness by rotary evaporation. All sp tra were taken in 0.5 M NaCl, pH 6.8 at 5° C unless otherwise noted. Some of the... 

Spectra/pore 3 dialysis tubing (3500 MW cutoff. Cat. No. 132725) is from Spectrum. The Centricon-30 concentrator (Cat. No. 4208) is purchased from Amicon, Inc. Tissue culture flasks (Corning, 75 cm. Cat. No. 10-126-41), plastic polystyrene centrifuge tubes (Corning, Cat. No. 05-526A for 15 ml and Cat. No. 05-538-49 for 50 ml), and bottle filters (0.2 //m. Cat. No. 09-740-28E for 150 ml and 09-740-40A) are from Fisher Scientific. Cell-porator Electroporation System I and disposable individually sterilized electroporation chambers (Cat. No. 1600AA) are from Gibco-BRL. 

The Centricon filter must be prewashed prior to filtering the peptides from the DR complexes. A contaminant exists in the membrane that disrupts LC-MS analysis of the peptides. 

If the concentration of the affinity-purified antibody is low, it is recommended that they be concentrated to approx 0.5-2 mg/mL with a 30-kDa Centricon-filter apparatus prior to storage. Otherwise, include BSA to 1 mg/ml to reduce loss of the antibody by absorption to the walls of the storage tube Purified antibodies should be aliquoted into 5- to lO-pL aliquots in SOO-pL microcentrifuge tubes to minimize repeat freeze-thawing and stored at -80°C. Sodium azide can be added to the thawed antibodies (final concentration 0.01%) to maintain storage at 4°C. 

The release of blue dextran from the microspheres was studied in 25 mM buffer phosphate solution (pH 7.5) incubated in a rotatory shaker at 200 rpm and 37 C. Biocatalytic release of azo-BSA and sulfanilic acid from the microspheres was performed with subtilisin or lipase in 25 mM Tris-CIH buffer solution (pH=7.8) incubated at 200 rpm and 37°C. Azo-BSA and sulfanilic acid were determined spectrophotometrically at 334 nm. The presence of sulfanilic acid in the supernatant was assayed after precipitation of azo-BSA with 5% TCA for 15 minutes at 0°C, followed by centrifugation (10,000 xg, 20 minutes at 4°C). Controls without enzymes, and with protease previously inhibited with 1.0 mM diisopropyl fluorophosphate, or thermal inactivate lipase (heated at 100 C) were included. Chemical release of BSA from the microspheres was performed by incubating the microspheres in a buffer phosphate (100 mM, pH=7.4) until total microsphere disintegration. For CD analysis of BSA, samples were filtered through 100 kDa. MWCO devices (Centricon, Millipore, Billerica, MA, USA). 

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