Amersham Pharmacia Biotech

FIGURE 2.1 SEC analysis of a polymeric dextran sample (Mw = 1000) on Superdex peptide HR 10/50. The very high resolution between individual components of the sample is obtained by using two columns in series. Courtesy of T. Andersson. (Reproduced with permission from Amersham Pharmacia Biotech.)... 

FIGURE 2.3 Selectivity curves of various modem SEC media. (I) Superdex peptide, (2) Superdex 7S, (3) Superdex 200, (4) Superose 12. (S) Superose 6, (6) Sephacryl S-lOO HR. (7) Sephacryl S-200 HR, (8) Sephacryl S-300 HR, and (9) Sephacryl S-400 HR. (Reproduced with permission of Amersham Pharmacia Biotech.)... 

FIGURE 2.9 Industrial fractionation by SEC using Sephacryl S-lOO in three BPSS columns (L = 30 cm, i.d. = 140 cm) to give a total bed volume of 1500 liters. Courtesy of R. Hersberg. (Reproduced by permission of Amersham Pharmacia Biotech.)... 

Four column systems are available from Amersham Pharmacia Biotech that can be used to pack SEC media for various applications at the laboratory scale. These include C, XK, SR, and HR column systems. All of the laboratory-scale columns are constructed with borosilicate glass tubes. Columns for larger scale process applications include INdEX, BPG, EineLINE, BPSS, and Stack columns. The larger scale columns are constructed to meet stringent validation requirements for the production of biopharmaceuticals. Each of the column types are described. 

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

MegaBACE is commercially available from Amersham Pharmacia Biotech (Freiburg, Germany). 

Immunodetection is performed by chemiluminescence (ECL , Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). 

Approximately 1 mg of total protein is pre-incubated with 25 /il of A-Sepharose beads CL-4B (Amersham Pharmacia Biotech) in 300 /d of binding buffer FLA for 1 h at 4° with gende rocking. The sample is then spun down at 1000 rpm for 3 min in an Eppendorf F 45-24-11 rotor, preserving the supernatant for further use. An aliquot of 3% of the total protein from each reaction is stored at —20° to provide the input reference sample in the subsequent analysis. 

Amersham Pharmacia Biotech. Bjorkgatan 30 SE-751 84 Uppsala Sweden... 

Fig. 3. Commercially available carbocyanine dyes with reactive groups for fluorescence labeUing pm poses. Cy3 (absorption maximum 550 nm, fluorescence maximum 570 nm), Cy5 (650 nm/670 nm), Cy7 (743 nm/767 nm) and Cy5.5 (675 nm/694 nm) Amersham Pharmacia Biotech, Freiburg, Germany...

Osborn, J. A review of radioactive and non-radioactive-based techniques used in life science applications. Part I Blotting techniques. Life Science News, Amersham Pharmacia Biotech 2000, 6,1-6. 

Maximum reproducibility of 2-DE protein patterns in large-scale proteome analysis can be achieved by simultaneous electrophoresis of SDS-PAGE. Amersham Pharmacia Biotech (Piscataway, NJ) firm developed a multiple vertical second-dimension... 

Figure 5-42 Intact DNA from the chromosomes of three strains of the malaria parasite Plasmodium falciparum, ranging from 750 Kb to 5 Mb, separated by pulsed-field gel electrophoresis. Courtesy of C. Smith and T. E. Wellems. Reproduced by permission of Amersham Pharmacia Biotech Inc.

Figure 5-49 A DNA sequencing gel obtained using a segment of DNA from salmon sperm selected by suitable oligonucleotide primers, amplified by PCR, and sequenced with a 35S label in the primer. Four samples were used, one with each of the four dideoxy chain terminators (A, G, C, T, A, C, G, T from left to right). After electrophoresis the shorter fragments are at the lower end of the gel. The sequence of the strand complementary to the template strand whose sequence is being determined is read from the bottom of the gel. Here it starts CTATGATAC. Reproduced by permission of Amersham Pharmacia Biotech, Limited.

Figure 26-7 Genetic map of cloning plasmids pBR322 and pUC18. Abbreviations ori, origin of replication Amp1, ampicillin resistance gene Tet1, tetracycline resistance gene. Other abbreviations are for sites cleaved by specific restriction endonucleases, a few of which are defined in Table 26-2. The nucleotide sequence numbers and directions of transcription are also indicated. Reproduced by permission of Amersham Pharmacia Biotech Inc.

Side-outlet gradient maker for linear gradients (e.g., Hoefer SG50 for standard gels and Hoefer SGI5 for mini-format gels Amersham Pharmacia Biotech) Peristaltic pump capable of delivering 1 to 6 ml/min... 

Flatbed electrophoresis apparatus (e.g., Multiphor II Amersham Pharmacia Biotech)... 

Vacuum gel dryer (e.g., Hoefer GD2000 Amersham Pharmacia Biotech) attached to vacuum pump or air gel dryer with dryer with drying frames (e.g., Hoefer Easy Breeze Amersham Pharmacia Biotech)... 

Dissolve 5.04 g urea (final 8 M) and 788 (J.1 carrier ampholyte mixture (e.g., Pharmalyte Amersham Pharmacia Biotech final 7.5%) in water to 10.5 ml. Add a reducing agent (60 mM dithiothreitol) and/or a detergent (0.5% [w/v] Triton X-100, CHAPS, or octyl glucoside), if desired. Make fresh. 

Contributed by Tom Berkelman Amersham Pharmacia Biotech San Francisco, California... 

Dr. Berkelman wishes to acknowledge Amersham Pharmacia Biotech scientists, particularly Reiner Westermeier and Nancy Laird, for providing additional material. 

Contributed by Alan Williams Amersham Pharmacia Biotech Piscataway, New Jersey... 

Betalains Materials Solvent A 18/82(v/v) CH3OH/0.05 M KH2PO, adjust to pH 2.75 with H3P04 Solvent B. CH3OH Sephadex G-25 (Amersham Pharmacia Biotech) water slurry, pH 2.0 adjust pH with HC1 Sample of beet juice or beet tissue extract (see Support Protocol), pH 2.0 adjust pH with HC1 1% acetic acid Four 7.88-mm x 61-cm Bondapak C,8/Porasil B columns connected in series 17.8/81.2/1.0 (v/v/v) CH3OH/0.05 M KH2P04/acetic acid 0.1% HC1... 

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