Centricon

Total polysaccharides were recovered in the ultrafiltration retentates on a Carbosep M5 membrane (Tech-Sep, MWCO 20 kDa) in the case of the red wine, or on a Centricon 30 membrane (Amicon, MWCO 30 kDa) in the ease of the apple and tomato juices. RG-II purification from the total polysaccharide coneentrates included, if necessary, several chromatography steps ... 

Another way to detect small molecules in the final formulated protein product without the interference from the protein signals is to remove the protein by ultrafiltration. Figure 12.4 compares a section of the proton NMR spectra of a biopharmaceutical protein product before (upper spectrum) and after (bottom spectrum) the protein was removed by ultrafiltering the sample with a Centricon-10 (Millipore Corp, Bedford, MA). Removing protein results in a flatter baseline (bottom spectrum). If small molecules are present in a protein sample, the removal of the protein may allow for unobstructed detection of the small molecules. In this case, a small amount of acetate ( 1 pg/rnl) is detected in the sample [bottom trace, Figure 12.4], Figure 12.5 shows that spikes of 10 p.g/ml of acetate and MES into the protein sample are fully recovered after the ultrafiltration to remove the protein. This example demonstrates that the interference of protein with the detection and quantitation of small-molecule impurities in a formulated protein product can be effectively eliminated by ultrafiltration. 

Protein removed by filtration using Centricon-10 membrane. 

Figure 12.4 A section of the proton NMR spectra of a biopharmaceutical protein product before (upper spectrum) and after (bottom spectrum) the protein was removed by ultrafiltering the sample with Centricon-10.

In general, a threefold dilution of the injected sample volume is to be expected. If necessary, the antibodies can be concentrated after this step. This can be conveniently accomplished using Centricon centrifuge concentrators (Amicon). 

An F/P ratio of two to five is optimal, since ratios below this yield low signals, whereas higher ratios show high background. If the F/P ratios are too low, repeat the coupling reaction using fresh fluorochrome solution. The IgG solution needs to be concentrated prior to reconjugation (e.g., Centricon-30 microconcentrator from Amicon Co., Beverly, MA, can be used to concentrate the IgG solntion). 

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. 

If the protein concentration of the eluate is too low for further investigation, concentrate the sample using a centrifuge membrane concentrator (Centricon) with molar mass cut-off significantly lower than the Mr of the analyzed protein or by precipitation with TCA or acetone. 

Concentrate the toxin to 10 mg/ml using a centrifugal concentrator with a molecular weight cutoff of 10,000 (Amicon Centricon 10 concentrators work well for this purpose). Retain this solution for the conjugation reaction. 

Remove excess fluorophore from the labeled oligo using gel filtration on a column of Sephadex G-25, dialysis, or a Centricon centrifugal concentrator. 

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

Put 1 mL of protein synthesis solution into Centricon YM-3 and centrifuge (5000g) at 4°C. 

Alternatively, buffer exchange can be done by repeated dilution with NMR sample buffer and concentration using Centricon YM-3. For example, four repeats of fivefold dilution and concentration with NMR sample are sufficient for buffer exchange. 

For the larger dendrimers incorporating an arene 9 and adamantyl core 10, preliminary membrane filtration experiments showed quantitative retention of the dendrimer-rhodium complexes using a Millipore Centricon-3 membrane and methanol as the solvent [53]. Results on hydrogenation reactions using these dendrimer catalysts in a CFMR have not yet been reported. 

Cap the tube with the Centricon retentate cup, then backspin the retentate (about 40 /zl) into the cup in a swinging-bucket rotor by bringing the speed to 1000 rpm and then braking immediately. The retentate contains the biotinylated dsDNA for use in the capture step below. 

If desired, 100-250 pi of the liquid in the Centricon filtrate cup may be concentrated by evaporation, and selective elimination of primers can be evaluated on an agarose gel. 

Centricon tubes may be rinsed and reused for the same preparations later in the procedure. Fill the top of the tube (Centricon concentrator) with sterile water, cap with the retentate cup, and shake gently. Repeat a total of 3 times. The tube need not be dried, but the retentate cup must be pipetted dry before reusing it to capture the final ssDNA, so that it will not be diluted. Rinsing can conveniently be done during incubation in the capture step, below. 

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. 

After the 6-min incubation, remove the NaOH solution (which contains the released, nonbiotinylated ssDNA strand) from the magnetic beads and add it to the ammonium acetate in the Centricon-100 tube. It is very important not to pipette any magnetic beads into the NaOH fraction. Most ssDNA will be recovered in this single NaOH wash. If desired, Steps 2 and 3 can be repeated. 

Add sterile water to the neutralized ssDNA solution in the Centricon-100 tube to a total volume of 2.0 ml. Spin for 25 min in a fixed-angle rotor at the highest speed recommended for the Centricon tubes. Add... 

The following procedure reduces the cost and time requirements for ssDNA preparation and yields enough template for one or two sequencing reactions. It is designed to speed analysis of a short DNA segment from many different individuals in a population. The PCR reaction is performed in a smaller volume, the Centricon prespin is omitted, and concentration steps are replaced by 2-propanol precipitation. 

A DNA extraction protocol that has proved useful for most ancient tissues is a modification of the protocol initially published by Blin and Stafford.20 Approximately 0.1 g of small pieces of soft tissue is added to 5 ml of extraction buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl, 1% (w/v) sodium dodecyl sulfate (SDS), 10 mg/ml dithiothreitol (DTT), and 0.5 mg/ml proteinase K. Incubation at 37° with gentle agitation overnight will allow most or all of the tissue to go into solution. An equal volume of phenol, equilibrated with 1 M Tris-HCl (pH 8.0), is added. When the phenol is being equilibrated, care should be taken to use uncontaminated Tris buffer and to measure the pH only on aliquots that are removed from the water phase and then discarded. Two phenol extractions and one chloroform extraction are performed, and the water phase is concentrated and purified on a Centricon 30 microconcentrator (Amicon, Danvers, MA). The reten-tate can be stored frozen, preferably in a few aliquots. In all cases solutions should be manipulated with DNA-free positive displacement pipettes. 

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 products of first-strand cDNA synthesis are treated with RNase A to remove excess mRNA and extracted with phenol to remove enzymes. Excess nucleotides and salts are removed by ultrafiltration in a Centricon 30 tube (Amicon, Danvers, MA). 

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

The complex between DnaK and RCMLA was formed by incubating equ volumes of both protein stock solutions for about 100 min at 37t C 500 pL of the reaction mixture was injected and several 1.5-mL fractions were collected and concentrated using a Centricon 3 (cut-off 3000 Da) fraction 4 corresponded to the peak attributed to the DnaK-RCMLA complex (elution volume ca. 16 mL. see Fig. 1). The final volume of the concentrated fractions was about 150 pL of which 10 pL was treated with SDS sample buffer, heated at 95°C for 2 min and loaded (1 pL) onto a SDS-PAGE gel. After developing with freshly made Coomassle Blue R solution, the gel was dried overnight at 37°C and then scanned with the densitometer. Only two bands, corresponding to the positions of standard RCMLA and DnaK samples, were observed for fraction 4. The intensity of the... 

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