Dynal

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment). (See text for full caption). 

Magnetic Dynabeads with M280 sheep antibody to mouse immunoglobulin G (IgG Dynal Biotech/Invitrogen). 

Immunomagnetic beads Dynabeads M-450 coated with sheep antimouse IgG, sheep or rat antimouse IgG subclass (Fc-specific), or preconjugated with MAbs specific for CD4 and CD8 (available from Dynal, Wirral, Merseyside, UK) A broad range of other preconjugated beads are now supplied by Dynal and are summarized m Note 2. They will remain stable for up to 1 yr when stored at 2-8°C Thiomersal (0 01% [w/v]), or sodium azide (0.02% [w/v]) is used as preservative. Hazard warning Both sodium azide and thiomersal are irritants to the skm and eyes, and are toxic if inhaled or ingested. Handle with care. 

Permanent magnet Dynal Magnetic Particle Concentrator (MPC-1, MPC-2, MPC-6, or MPC-Q) (Dynal, UK), or equivalent magnet (e g., from Biolab, Belgium). 

Detachabead is available from Dynal for positive selection of CD4+, CD8+, CD 19+, and CD34+ cell subsets (23-30) This reagent has also been used to select positively for CD45+ cells (31), HLA-DR+ Langerhans cells (32), and c-kit+ human lung mast cells (33) However, its suitability for dissociation of beads coated with MAbs of other specificities should always be assessed by the user in a pilot study. 

Dynal Ltd. (1996) Cell separation and protein purification, in Technical Handbook, 2nd ed. 

Streptavidin-coupled M-280 Dynabeads (Dynal, Oslo, Norway). 

Sample mixer with 12-tube mixing wheel and a magnet (Dynal Biotech). 

Tosylactivated magnetic beads (MB-Tosyl) (Dynabeads M-280 Prod. No. 142.03, Dynal Biotech ASA, Oslo, Norway). Hydroquinone (Sigma), hydrogen peroxide (Merck, Germany). Bradford solution (Bio-Rad protein assay Catalog No. 500-0006) was purchased from Bio-Rad laboratories GMGH (Munich, Germany). 

Tris(hydroxymethyl)methylamine (TRIS), sodium chloride, sodium citrate, ethylenediamine tetraacetic acid disodium salt (EDTA), lithium chloride, Tween 20, streptavidin 10 nm colloidal gold labelled, hydrochloric acid (37%), nitric acid, streptavidin-coated paramagnetic beads (MB) with a diameter of 2.8 pm, Dynabeads M-280 Streptavidin (Dynal Biotech, Oslo, Norway) biotinylated probe oligonucleotides which sequences are shown in Table 53.1. 

Dynabead Dynal Biotech, Oslo, Norway 4.5 Monoclonal antibodies www.dynalbiotech.com... 

Yet another approach for mRNA isolation is the use of oligo(dT)-coated magnetic beads for mRNA isolation (Figure 7.5). Such beads with attached oligo(dT) are available commercially (Dynal, Lake Success, NY). Alternatively, the oligonucleotide can be chemically attached to magnetic beads [29]. 

Amersham-Pharmacia (Piscataway, NJ) Dynal (Lake Success, NY)... 

Various methods are available for fractionating lymphocytes to obtain pure cultures of the different sorts of white blood cells (Johnstone and Thorpe, 1987). A particularly simple method involves the use of magnetisable polymer particles precoated with monoclonal antibodies (Dynal Ltd. Appendix 3). 

Dynal (U.K.) Ltd., Station House, 26 Grove St., Wirral, Merseyside L62 2AB. Dynatech Labs Ltd. (see Gibco). 

Sheep antimouse IgG-coatedmagnetic beads (Dynabeads M450, Dynal 11001). 

The 5 mL tube is placed in a magnetic field (DYNAL MDC 1) for 2 min, the supernatant is gently removed with a 1 mL disposable polypropylene transfer pipette. Resuspend the beads in 2 mL HBSS/1% BSA and place the tube in the magnetic field again for 2 min. Collect the supernatant, combine the 2 supernatants and centrifuge at 300,g for 10 min. Resuspend the cells in the desired volume of HACM. 

Coupling proteins (by covalent methods) to magnetic supports is the most common approach (105). Various classes of proteins, such as lectins (110) and antibodies (111), and a large number of enzymes including invertase (112,113), papain (106, 114) a-chymotrypsin (114, 115), P-galactosidase (114), adenylate kinase (112), acetate kinase (112), and horseradish peroxidase (112), have been immobilized. In fact, such supports are now commercially available, for example, Enzacryl FEO-M (available from Aldrich Chemictd Company, USA) and Dyna (sold by Dynal, Norway). 

Immunomagnetic Beads Dynabeads uncoated, or coated as M-280 or M-450 sheep antimouse IgG (available from Dynal, Wirral, Merseyside, UK). Stored at 2-8°C, they will remain stable for up to 1 yr. Thiomerosal... 

Permanent Magnet Dynal Magnetic Particle Ck>ncentrator (MC1 or MPC 6) (Dynal, UK) or equivalent magnet (Biolab, Belgium also supply magnets). 

Immunomagnetic beads can be obtained from Dynal, UK with sheep andmouse IgG attached (Secdon 3.1.1.) or uncoated for physical absorpdon of IgM andbodies or chemical coupling of IgG andbodies (as recommended by the producers) (Section 3.1.2.). 

Aqueous two-step swelling is a technique for producing monodisperse polymer particles over a size range much greater than that achievable by direct dispersion polymerisation. The properties of the particles, such as porosity, can also be controlled. The concept was first introduced by Ugelstadt and Mork [28] and has since been adopted by many workers. It is used to produce a number of commercial products for chromatography and bioseparations, such as Mono-beads by Pharmacia and Dynabeads by Dynal. 

The basic magnetic-bead patent appears to be the property of Dynal, but Clemente Associates (formerly Quantum Magnetics, Madison, CT) produces Ni-bearing magnetic particles of 3- to 5-pm diameter for... 

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