Dynabeads® Streptavidin Trial Kit（二）

3. General Immobilization Protocol

            Wash the Dynabeads® before use.

                  a)      Add the biotinylated molecule to the washed Dynabeads® .

                  b)      Incubate for 15-30 min at room temperature with gentle rotation of the tube.

                  c)      Place the tube in a magnet for 2-3 mins and discard the supernatant.

                  d)     Wash the coated beads 3-4 times in washing buffer.

                  e)      Resuspend to desired concentration in a suitable buffer for your downstream use.

            Here are some examples of immobilization protocols for specific applications.

            Immobilization of Nucleic Acids

                 f)       Resuspend beads in 2x B&W Buffer to a final concentration of 5 μg/μl (twice original volume).

                 g)      To immobilize, add an equal volume of the biotinylated DNA/RNA in H₂O to dilute the NaCl concentration in the 2x B&W Buffer from 2M to 1 M for optimal binding.

                 h)      Incubate for 15 mins at room temperature  using gentle rotation. Incubation time depends on the nucleic acid  length: short oligonucleotides (< 30 bases) require max. 10 mins. DNA  fragments up to 1 kb require 15 mins.

                  i)        Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins.

                  j)        Wash 2–3 times with a 1x B&W Buffer.

                 k)      Resuspend to the desired concentration.  Binding is now complete. Resuspend the beads with the immobilized  DNA/RNA fragment in a buffer with low salt concentration, suitable for  downstream applications.

            Immobilization of Antibodies/Proteins

                 l)        Incubate the beads and biotinylated  antibodies in PBS for 30 mins at room temperature using gentle rotation.

               m)    Separate the antibody-coated beads with a magnet for 2–3 mins.

                n)      Wash the coated beads 4–5 times in PBS containing 0.1% BSA.

                o)      Resuspend to the desired concentration for your application.

4. Release of Immobilized Biotinylated Molecules

The biotin-streptavidin bond is broken by harsh conditions. 5 mins  incubation at 65°C or 2 mins at 90°C in 10 mM EDTA pH 8.2 with 95%  formamide will typically dissociate >96% of immobilized biotinylated  DNA. Alternatively, boi the sample for 5 mins in 0.1% SDS for  dissociation. Please note that proteins will be denatured by such  treatmen and Dynabeads® Streptavidin can not be re-used. It has also  been reported that the biotin-streptavidin interaction can be broken by a  short incubation in nonionic aqueous solution at temperature above  70°C.

5. Immunoassay Strategies

Due to their high surface area per weight, uniformity, excellent  batch reproducibility and ease of adaptation to automated processes,  Dynabeads® have become the solid phase of choice for developing  immunoassays

6. Automation

Magnetic separation and handling using Dynabeads® can easily be  automated on a wide variety of liquid handling platforms. Dynabeads®  MyOne are ideal for automation applications due to the small size, low  sedimentation rate and high magnetic mobility.

7. Technical Information

Binding capacity

Both the size of the molecule to be immobilized and the biotinylation  procedure will affect the binding capacity. Large as well as small  biotinylated molecules can be immobilized. The capacity for biotinylated  molecules depends on steric availability and charge interaction between  bead and molecule and between molecules. There are two or three biotin  binding sites available for each streptavidin molecule on the surface of  the bead after immobilization.

                         l  Optimize the quantity of beads used for each individual application by titration.

                         l  Use up to two-fold excess of the binding  capacity of the biotinylated molecule to saturate streptavidin.

                         l  Binding efficiency can be determined by comparing molecule concentration before and after coupling.

           
              Typical binding capacities for one mg of Dynabeads® .

*Oligonucleotides and DNA fragments For oligonucleotides, capacity is  inversely related to molecule size (number of bases). Reduced binding  capacity for large DNA fragments may be due to steric hindrance.