Dynabeads

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. 

An alternative approach for some human cell-surface markers (CD4, CD8, CD 19, and CD34) is the use of Detachabead, which is an anti-Fab polyclonal antiserum that causes dissociation of bound MAbs preconjugated to Dynabeads (see Note 9). For protocols detailing use of Detachabead see refs. 23—33. 

This alternative approach to IMS may therefore resolve some of the problems encountered using Dynabeads. 

Jackson, C. J, Garbett, P K., Nissen, B., and Schrieber, L (1990) Binding of human endothelium to Ulex europaeus I-coated Dynabeads. application to the isolation of microvascular endothelium J Cell Science 96,257—262. 

A new method for detachment of Dynabeads from positively selected B lymphocytes. J Immunol. Methods 146, 195-202. 

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

In this approach, the phage antibodies react with biotinylated antigen in solution, and the complex is then captured using Streptavidin coupled to Dynabeads. 

Block 1.5 mL (per selection) streptavidin M-280 Dynabeads by adding 5 mL 4% MPBS, and rotate in a 15-mL tube on an end-over-end mixer for 5 min at room temperature... 

Add 1 5 mL of blocked streptavidin Dynabeads to the phage bound to S-S biotinylated antigen... 

Repeat the selection procedure another three to four times (use a 1 -mL aliquot for selection, and store the remaining phage at 4°C). For the second and subsequent rounds of selection, mix 1 mL phage from the previous round of selection with 0.5 mL of 6% MPBS, 10 pL Tween-20, and biotinylated antigen to give a final concentration of 50 nM Use only 300 pL of blocked streptavidin-coated Dynabeads... 

In a microtube, add 1 mg of M-270 streptavidin-coated Dynabeads to 1 mL TBS and place the tube on a magnet to discard the supernatant. Off the magnet, resuspend the beads in 1 mL of MTBS for blocking nonspecific sites. Place the microtube on a rotating wheel for 1 h at room temperature. The rotation is necessary for keeping the beads in suspension and should be slow. 

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... 

QuickPrep Micro mRNA purification Dynabeads mRNA DIRECT Micro S.N.A.P. Total RNA Isolation Micro-FastTrack 2 mRNA SV Total RNA Isolation system PloyATtract system 1000 Rneasy Mini... 

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

MACS and FACS are used instead of magnetic sorting with Dynabeads. 

If further purification is required add antibody-coated (anti-CD2,-CD14, and -CD19) magnetic beads (Dynabeads M-450) at a ratio of 10 beads/cell. Incubate at 4°C for 30 min with gentle rocking. 

Fig. 5 Nucleic acid immobilization onto (a) silica 60 nm particles, (b) amino terminated 0.31 xm latex beads and (c) poly(T) modified Dynabeads . MPTS 3-mercapto-propyltrimethoxysilane. (

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... 

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. 

P20/I55.3 All foods Detection of Escherichia coli 0157 AFNOR-DYN-16/1 -03/96 Dynabeads anti Escherichia coli 0157... 

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