FPLC

Fig. 7. Chromatograms of size-exclusion separation of IgM (mol wt = 800,000) from albumin (69,000) where A—D correspond to IgM aggregates, IgM, monomer units, and albumin, respectively, using (a) FPLC Superose 6 in a 1 x 30 — cm long column, and (b) Sepharose CL-6B in a 37-cm column.

Ion Exchange Chromatography - Basic principles of ion exchange chromatography and studies conducted from Texas A M University. http //ntri.tamuk.edu/fplc/ion.html. 

Sephadex, Sepharose, Sephacryl, Superose, Superdex, FPLC, SMART, HiTrap, HiPrep, and INdEX are trademarks owned by Amersham Pharmacia Biotech AB. 

Thirty-two years later, Ko jima etal. (2000a) purified Latia luciferase by the following steps ammonium sulfate fractionation, gel-filtration, affinity chromatography and Mono-Q anion-exchange FPLC. 

The molecular masses of polygalacturonase and exopolygalacturonase were approximately determined by gel chromatography on Superose 12 using FPLC device (Pharmacia, Sweden) and the Calibration proteins II kit (Boehringer-Mannheim, Germany). 

Fig. 5. The approximate molecular mass determination of polygalacturonase [(0—0) - substrate 0.5% pectate, pH 4.6] and exopolygalacturonase [( — ) - substrate 1.0 pmol/ml of di(D-galactosiduronic) acid, pH 4.0] on Superose 12 column (FPLC device). Flow rate 0.5 ml/min. System 0.05 M phosphate buffer pH 7.0, 0.15 M NaCl. Standarts Ferritin (450 kDa), Katalase (240 kDa), Aldolase (158 kDa), Albumin (68 kDa), Albumin (45 kDa), Chymotrypsinogen A (25 kDa), Cytochrome C (12.5 kDa).

Anion exchange chromatography The reaction mixture is subjected to phenol/chloroform extraction to remove the T7 RNA polymerase using phenol equilibrated with 50 mM Na acetate (pH 4.5). After isopropanol precipitation, the pellets are resuspended in 20 mM MOPS buffer (pH 6.25) containing 350 mM NaCl. The excess unincorporated NTPs and the smaller abortive transcription products are removed by chromatography on anion exchange FPLC column (MonoQ 5/5 column, Amersham). 

The sample is loaded at a flow-rate of 1 ml/min onto the FPLC column equilibrated with the same MOPS buffer used to resuspend the RNA pellets. The free nucleotides are completely removed with a 5-ml wash with 350 mM NaCl and the RNA is eluted with a 20-ml (350—750 mM NaCl) linear gradient and analyzed by PAGE/urea gel electrophoresis (see later). Up to 2 mg of RNA can be loaded onto and eluted from a 1-ml (of resin) mono Q column without loss of resolution. The homogeneity of RNA in the fractions collected, as seen by gel electrophoresis, should be >90%. The appropriate fractions are pooled and the RNA collected by ethanol precipitation. The RNA pellet is washed twice with 70% ethanol, air-dried, and finally redissolved in DEPC-treated H20. The total recovery after the entire procedure of purification is = 90%. This protocol yields = 800 pmoles of purified 002 mRNA/pmole template DNA. 

FPLC system (Pharmacia, P-500 pumps, Frac-100 fraction collector, HR flow cell, UV-1 flow-through monitor, V-7 valve). 

Gel Filtration by Fast Protein Liquid Chromatography (FPLC)... 

Connect the Superose 6 column to the FPLC system (see Note 18), and equilibrate the column with 50 mL of BBS at a flow rate of 0.5 mL/min. Check the manufacturer s recommendations for optimal operating back pressures. 

In general, chromatography columns should not be left connected to pumps or to the UV monitors in salt solutions. Always include a wash step with water to remove any salt from the system. It is preferable to store the columns disconnected in 20% ethanol and to rinse the entire FPLC system, including pumps, tubing, and UV flow cell with water, followed by 20% ethanol. Keep a record of the column performance, and use it to determine when filter changes or columncleaning steps are required. 

FPLC components (two P 500 pumps, V7 injection valve, gradient controller, UV-1 detector, Frac-100 fraction collector, 50 mL Superloop [Pharmacia], dualchannel chart recorder, or similar components). 

FPLC Ion Exchange and Chromatofocusing—Principles and Methods. (1985) Pharmacia-LKB, Offsetcenter, Uppsala, Sweden. 

Figure 14.1 Protocols used to purify RNA according to their nucleotide length. Note that HPLC-FPLC can be used after PAGE to cleanse an RNA sample whatever the length of the RNA.

To a stirred soln of TBPI in 80% aq MeCN (0.75 mM) was added at rt H-Ile-Val-Gln-Cys-Arg-OH (1 equiv). After 6 h, H-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-OH (1 equiv) was added and the soln was kept at rt overnight. Upon completion of the reaction as monitored by RP-FPLC, the MeCN was removed under reduced pressure and the precipitated phthalimide was removed by filtration. The resulting... 

The column was equilibrated and initially eluted with 20 mM Tris-HCI (pH 7.6). Elution of the bound fraction was carried out by using 1 M NaCI in the equilibration buffer. All chromatographic steps were performed at the flow rate of 100 ml/h. Further separation selected fraction Q1, which was lyophilised and dissolved in 100 mM Tris-HCI (pH 7.6) buffer was performed onto a FPLC Superdex75 column at a flow rate 0.5 ml min. ... 

---