For rearch u only
INDEX
1. PRODUCT DESCRIPTION
2. PROTOCOLS
3. TECHNICAL INFORMATION
4. OTHER DYNABEADS STREPTAVIDIN PRODUCTS
5. REFERENCE
6. GENERAL INFORMATION
1. PRODUCT DESCRIPTION
1.1 Intended U
Dynabeads M-270 Streptavidin are ideal for nucleic acid diagnostics, specifically with samples with a hi
gh chaotropic salt concentration, immunoassays involving small biotinylated antigens and applications that are not compatible with BSA (the beads are not blocked with BSA).
1.2 Principle
Add Dynabeads to a sample containing biotinylated molecules such as peptides, oligonucle-otides etc. During a short incubation, the biotinylated molecule will bind to the beads. Separate the molecule-bead complex with a Dynal magnet. Capture, washing and detection can be optimized for manual or automated u. With indirect capture, mix the biotinylated molecule 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-pha kinetics.
1.3 Description of Materials
Dynabeads M-270 Streptavidin are uniform, superparamagnetic beads of 2.8 µm in diameter with a streptavidin monolayer covalently coupled to the hydrophilic bead surface. This layer ensures negligible streptavidin leakage while the lack of excess adsorbed Streptavidin ensures batch consistency and reproducibility of results.
Materials Supplied
Dynabeads M-270 Streptavidin are supplied as a suspension containing 10 mg (6-7 x 108) Dynabeads per ml, dissolved in phosphate buffered saline (PBS) pH 7.4, containing 0.09% NaN3as prervatives. Available in three volumes:
2 ml ( 653.05)
10 ml (Cat. no. 653.06)
100 ml (Cat. no. 653.07).
Additional Materials Required
–Magnet for manual or automated protocols. See /magnets-lection for recommendations.
–Mixing device with tilting and rotation.
–Buffers and Solutions (e Table 1).
–Biotinylated compounds. For advice on biotinylation, e dynal.Section4.2 of "The Handbook (/handbook) gives a guide to available biotinylation reagents.
Table 1: Recommended buffers and solutions The salt concentration and pH (typically 5-9) of the chon binding/washing buffers can be varied depending on the type of molecule to be immobilized. Beads with immobilized mole-cules are stable in common buffers.
2. PROTOCOLS
�CRITICAL NOTES
•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, pa-ration is often complete in under 1 min, once you can e the beads collected.
•For diluted sample increa the incubation time or isolate in smaller batches using the same beads in each batch.
•U a mixer to tilt/rotate the tubes so Dynabeads do not ttle at the tube bottom.•Avoid air bubbles
during pipetting.
•Free biotin in the sample will reduce the binding capacity of the beads. A disposable paration 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. PCR Clean Up products are available from
2.1 Immobilization Procedure
2.1.1 Bead Preparation
1.Resuspend the beads in the original vial by rotation or vortexing.
2.Calculate the amount of beads required bad on their binding capacity, e table 3, and transfer the beads to a new tube.
3.Wash Dynabeads (e 2.1.2) to remove prervatives.
Recommended washing buffers:
– nucleic acid applications: 1x B&W Buffer
– antibody/protein applications: PBS, pH 7.4
Note:For many applications it can be an advantage to add a 0.01-0.1% Tween®20 to the washing/binding buffers to reduce non-specific binding.
Table 2: Typical binding capacities for one mg of Dynabeads.
*) Oligonucleotides and DNA fragments
For oligonucleotides, capacity is inverly related to molecule size (number of bas). Reduced binding capacity for large DNA fragments may be due to steric hindrance.
2.1.2 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 sup-plied in RNa-free solutions, perform the following steps after washing for RNA applications: 9.Wash the beads twice in Solution A for 2 mins. U the same volume of beads as re-
commended in step 7.
10.Wash the beads once in Solution B. U 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.
Cat. no.653.05
653.06
Rev. no.006 Dynabeads®M-270 Streptavidin
For coupling of nucleic For Dynabeads treatment acids before RNA manipulations Binding and washing Solution A:
(B&W) Buffer (2x):DEPC-treated 0.1 M NaOH,
10 mM Tris-HCl (pH 7.5)DEPC-treated 0.05 M NaCl
1 mM EDTA
2 M NaCl Solution B:
DEPC-treated 0.1 M NaCl Free Biotin [pmol]> 950 Biotinylated peptides [pmol]~ 200 Biotinylated antibody [µg]up to 10 ds DNA [µg] *)~ 10
ss oligonucleotides [pmol] *)~ 200
007
that such transfer is not for any Commercial Purpo, and that such collaborator agrees in writ-ing (a) not to transfer such materials to any third party, and (b) to u such transferred materials and/or information solely for rearch and not for Commercial Purpos. Commercial Purpos means any activity by a party for consideration and may include, but is not limited to: (1) u of the pro-duct or its components in manufacturing; (2) u of the product or its components to provide a r-vice, information, or data; (3) u of the product or its components for therapeutic, diagnostic or prophylactic purpos; or (4) resale of the product or its components, whether or not such product or its components are resold for u in rearch.Invitrogen Corporation will not asrt a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product bad upon the manufac-ture, u or sale of a therapeutic, clinical diagnos-tic, vaccine or prophylactic product developed in rearch by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was ud in the manufacture of such product. If the purchar is not willing to accept the limitations of this limi-ted u statement, Invitrogen is willing to accept return of the product with a full refund. For infor-mation on purchasing a licen to this product for purpos other than rearch, contact Licensing Department,Invitrogen Corporation,
1600 Faraday Avenue, Carlsbad,California 92008.
Phone (760) 603-7200.Fax (760) 602-6500.
Email:
6.6 Warranty
The products are warranted to the original purchar only to conform to the quantity and contents stated on the vial and outer labels for the duration of the stated shelf life. Invitrogen Dynal's obligation and the purchar's exclusive remedy under this war-ranty is limited either to replacement, at Invitrogen Dynal's expen, of any products which shall be defective in manufacture, and which shall be retur-ned to Invitrogen Dynal, transportation prepaid, or at Invitrogen Dynal's option, refund of the purcha price.
Claims for merchandi damaged in transit must be submitted to the carrier .
This warranty shall not apply to any products which shall have been altered outside Invitrogen Dynal, nor shall it apply to any products which have been subjected to misu or mishandling.ALL OTHER WARRANTIES, EXPRESSED, IMPLIED OR STATUTORY , ARE HEREBY SPECIFICALLY EX-CLUDED, INCLUDING BUT NOT LIMITED TO WAR-RANTIES OF MERCHANTABILITY OR FITNE
SS FOR A PARTICULAR PURPOSE. Invitrogen Dynal's maxi-mum liability is limited in all events to the price of the products sold by Invitrogen Dynal. IN NO EVENT SHALL INVITROGEN DYNAL BE LIABLE FOR ANY SPECIAL, INCIDENTAL OR CONSEQUEN-TIAL DAMAGES. Some states do not allow limits on warranties, or on remedies for breach in certain transactions. In such states, the limits t forth above may not apply.
2.1.3 General Immobilization Protocol
Wash the Dynabeads according to ction 2.1.2 before u.1.Add the biotinylated molecule to the washed Dynabeads.
2.Incubate for 15-30 min at room temperature with gentle rotation of the tube.
3.Place the tube in a magnet for 2-3 mins and discard the supernatant.
4.Wash the coated beads 3-4 times in washing buffer .
5.Resuspend to desired concentration in a suitable buffer for your downstream u.Here are some examples of immobilization protocols for specific applications.
2.1.4 Immobilization of Nucleic Acids
1.Resuspend beads in 2x B&W Buffer to a final concentration of 5 µg/µl (twice original volume).
2.To immobilize, add an equal volume of the biotinylated DNA/RNA in H 2O to dilute the NaCl concentration in the 2x B&W Buffer from 2M to 1 M for optimal binding.
3.Incubate for 15 mins at room temperature using gentle rotation. Incubation time de-pends on the nucleic acid length: short oligonucleotides (< 30 bas) require max. 10 mins.DNA fragments up to 1 kb require 15 mins.
4.Separate the biotinylated DNA/RNA coated beads with a magnet for 2-3 mins.
5.Wash 2–3 times with a 1x B&W Buffer .
6.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.2.2 Relea of Immobilized Biotinylated Molecules
The biotin-streptavidin bond is broken by harsh conditions. 5 mins incubation at 65°C or 2mins at 90°C in 10 mM EDTA pH 8.2 with 95% formamide will typically dissociate >96% of immobilized biotinylated DNA. Alternatively, boil the sample for 5 mins in 0.1% SDS for protein dissociation.
Plea note that proteins will be denatured by such treatment and Dynabeads Streptavidin cannot be re-ud.
It has also been reported that the biotin-streptavidin interaction can be broken by a short incubation in nonionic water at a temperature above 70°C (ref 1).
2.3 Immunoassay Strategies
Due to their high surface area per weight, uniformity, excellent batch reproducibility and ea of adaptation to automated process, Dynabeads have become the solid pha of choice for developing immunoassays (/IVD).
2.4 Automation
Magnetic paration and handling using Dynabeads can easily be automated on a wide variety of liquid handling platforms. Dynabeads MyOne™ Streptavidin C1 share similar pro-perties to Dynabeads M-270 Streptavidin but are smaller , making them ideal for automation applications due to their small size, low dimentation rate and high magnetic mobility.Selected protocols are available at /automation.
3. 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.•Optimize the quantity of beads ud for each individual application by titration.
•U up to two-fold excess of the binding capacity of the biotinylated molecule to saturate streptavidin.
•Binding efficiency can be determined by comparing molecule concentration before and after coupling.
4. OTHER STREPTAVIDIN DYNABEADS Bead type Available product formats
Dynabeads ®M-280 2 ml (Cat. No. 112.05D)Streptavidin 10 ml (Cat. No. 112.06D)
100 ml (Cat. No. 602.10)
Dynabeads ®MyOne™ 2 ml (Cat. No. 650.01)Streptavidin C1 10 ml (Cat. No. 650.02)
100 ml (Cat. No. 650.03)
Dynabeads ®MyOne™ 2 ml (Cat. No. 656.01)Streptavidin T1 10 ml (Cat. No. 656.02)
100 ml (Cat. No. 656.03)
Dynabeads ®Kit (Cat. No. 601.01)kilobaBINDER™
(For biotinylated DNA fragments > 2 kb)
5. REFERENCE
1. Holmberg et al.(2005) The biotin-Streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26, 501-510.For a list of lected references where Dynabeads M-270 have been ud, plea visit /streptavidin.
6. GENERAL INFORMATION
Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:200
3.
6.1 Storage and Stability
If stored unopened at 2–8°C, the Dynabeads are stable until the expiration date stated on the label.Store the vial upright to keep beads in liquid sus-pension, as drying of the beads will result in re-duced performance. Do not freeze the product.Thoroughly resuspend the Dynabeads in the vial prior to u. Dynabeads Streptavidin are not sup-plied in RNa free solution. Avoid bacterial conta-mination of the beads.
6.2 Warnings & Limitations
This product is for rearch u only. Not intended for any animal or human therapeutic or diagnostic u unless otherwi stated. Sodium azide is toxic if ingested. Avoid pipetting by mouth. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide buildup.
Certificate of Analysis /Compliance is available upon request.
Material Safety Data Sheet (MSDS) is available at
6.3 Trademarks & Patents
Dynal ®, Dynabeads ®and MyOne™are either regis-tered trademarks or trademarks of Invitrogen Dynal AS, Oslo, Norway. Any registration or trade-mark symbols ud herein denote the registration status of trademarks in the United States.Trademarks may or may not be registered in other countries.
Tween® is a registered trademark of ICI Americas Inc.
6.4 Intellectual Property Disclaimer
Invitrogen Dynal will not be responsible for viola-tions or patent infringements that may occur with the u of our products.
6.5 Limited U Label Licen
No. 5: Invitrogen Technology – The purcha of this product conveys to the buyer the non-transfer-able right to u the purchad amount of the pro-duct and components of the product in rearch conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot ll or otherwi transfer (a) this product (b) its components or (c) materials made using this pro-duct or i
ts components to a third party or other-wi u this product or its components or materials made using this product or its components for Commercial Purpos. The buyer may transfer in-formation or materials made through the u of this product to a scientific collaborator , provided
Invitrogen Dynal is a part of the Invitrogen Group.Contact details for your local Invitrogen sales office/technical support can be found at /contact
© Copyright 2007Invitrogen Dynal AS, Oslo, Norway.All rights rerved.
Revid:10.2007Printed:10.2007
SPEC-05661
