实验概要
Dynabeads® Streptavidin are ideal for numerous applications, including purification of proteins, nucleic acids purification, protein interaction studies, immunoprecipitation, immunoassays, phage display, biopanning, drug screening and cell isolation.
实验原理
Add Dynabeads® to a sample containing biotinylated molecules. During a short incubation, the biotinylated molecule will bind to the beads. Separate the molecule-bead complex with a magnet. Capture, washing and detection can be optimized for manual or automated use. With indirect capture, the biotinylated molecule is mixed with the sample to capture the molecule-target complex before adding Dynabeads®. Indirect target capture can be advantageous when molecule-target kinetics are slow, affinity is weak, molecule concentration is low or molecule-target binding requires optimal molecule orientation and true liquid-phase kinetics.
主要试剂
1. Materials supplied in the Dynabeads® Streptavidin Trial Kit. Sodium azide (NaN3) is used as a preservative. Abbreviations: Volume (V) Phosphate buffered saline (PBS)
Dynabeads® | V [ml] | Concentration [mg/ml] | Supplied in | Bead diameter [μm] |
M-280 Streptavidin | 1 | 10 6-7x108 beads/ml) | PBS, pH 7.4 / 0.1% BSA / 0.02% NaN3 | 2.8 |
M-270 Streptavidin | 1 | 10 (6-7x108 beads/ml) | PBS, pH 7.4, / 0.09% NaN3 | 2.8 |
MyOne™ Streptavidin C1 | 1 | 10 (7-12x109 beads/ml) | PBS, pH 7.4 / 0.01% Tween®-20 / 0.09% NaN3 | 1.0 |
MyOne™ Streptavidin T1 | 1 | 10 (7-12 x 109 beads/ml) | PBS, pH 7.4 / 0.1% BSA / 0.02% NaN3 | 1.0 |
2. Additional Materials Required
(1)Magnet (Dynal® MPC™) for manual or automated protocols. See www.invitrogen.com/magnets-selection for recommendations.
(2)Mixing device with tilting and rotation.
(3)Buffers and Solutions (see 3).
(4)Biotinylated compounds. For advice on biotinylation, see http://www.invitrogen.com/dynal. Section 4.2 of the handbook (http://probes.invitrogen.com/handbook) gives a guide available biotinylation reagents.
3. Recommended buffers and solutions:
For coupling of Nucleic Acids | For Dynabeads® treatment before RNA manipulations | For coupling of protein or other molecules |
Binding and washing (B&W)Buffer (2x): 1 mM EDTA 2 M NaCl | Solution A: DEPC-treated 0.1 M NaOH, DEPC-treated 0.05 M NaCl Solution B: DEPC-treated 0.1 M NaCl | PBS buffer pH 7.4 These buffers can also be used for your application if needed: PBS/BSA (PBS, pH 7.4 containing 0.1% (w/v) BSA) PBST (PBS, pH 7.4 containing 0.01% (v/v) Tween® 20) |
The salt concentration and pH (typically 5-9) of the chosen binding/washing buffers can be varied depending
on the type of molecule to be immobilized. Beads with immobilized molecules are stable in common buffers.
DEPC-treatment:
Add DEPC to a final concentration of 0.1% (1ml/L) to Solution A or B. Shake vigorously, incubate for 1 hour at room temperature. Ready to use after autoclaving.
Recipe for PBS buffer pH 7.4:
0.26 g NaH2PO4 x H2O (MW 137.99)
1.44 g Na2HPO4 x H2O (MW 177.99)
8.78 g NaCl (MW 58.5)
Dissolve in 900 ml distilled water, adjust pH if necessary and adjust to 1 liter.
实验步骤
1. Critical Notes
(1)In the protocols we recommend keeping the tube on the magnet for up to 2 mins to ensure that all the beads are collected on the tube wall. For non-viscous samples, separation is often complete in under 1 min, once you can see the beads collected.
(2)If you do not need to remove preservatives or change buffers you can omit Washing Procedure.
(3)For diluted sample or large sample volumes, increase the incubation time or isolate in smaller batches using the same beads in each batch.
(4)Use a mixer to tilt/rotate the tubes so Dynabeads® do not settle at the tube bottom.
(5)Avoid air bubbles during pipetting.
(6)Free biotin or biotinylated primer in the sample will reduce the binding capacity of the beads. A disposable separation column or a spin column will remove unincorporated biotin. Run the PCR with limiting concentrations of biotinylated primer, or remove free biotinylated primer by ultrafiltration, microdialysis or other clean-up protocols.
2. Immobilization Procedure
Bead Preparation
1) Resuspend the beads in the original vial by rotation or vortexing.
2) Calculate the amount of beads required based on their binding capacity, see table 3, and transfer the beads to a new tube.
3) Wash Dynabeads® to remove preservatives.
Recommended washing buffers:
l nucleic acid applications: 1x B&W Buffer
l antibody/protein applications: PBS, pH 7.4
Note: For many applications it can be an advantage to add a detergent e.g. 0.01-0.1% Tween® 20 to the washing/binding buffers to reduce non-specific binding.
Washing Procedure
4) Place the tube containing the beads on a magnet for 1-2 mins.
5) Remove the supernatant by aspiration with a pipette while the tube is on the magnet.
6) Remove the tube from the magnet.
7) Add washing buffer along the inside of the tube where the beads are collected and resuspend
(same volume of washing buffer as the initial volume of Dynabeads® taken from the vial or larger).
8) Repeat steps 4 to 7 twice, for a total of 3 washes.
If using Dynabeads® for RNA Manipulation:
As Dynabeads® Streptavidin are NOT supplied in RNase-free solutions, perform the following steps after washing for RNA applications:
9) Wash the beads twice in Solution A for 2 mins. Use the same volume of beads as recommended in step 7.
10) Wash the beads once in Solution B. Use the same volume of beads as in step 9.
11) Resuspend the beads in Solution B.
The beads are now ready to be coated with the biotinylated molecule of your choice.
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