实验概要
The Dynabeads and buffers provided in this kit will enable you to covalently immobilize antibodies (or other protein ligands such as lections, functional enzymes, etc.) of your choice onto the surface of Dynabeads. Antibody-coupled Dynabeads can then be used for downstream assays and experiments. Once the coupling reaction has been completed the resulting Dynabeads surface will exhibit ultra-low background binding. It is therefore not necessary to block the bead surface prior to use. For best results, please read through this material carefully prior to start.
实验原理
Antibodies (Ab) (or other protein ligands) of your choice are covalently coupled to the provided Dynabeads. Once antibodies have been coupled to the Dynabeads, the antibody coupled Dynabeads may then be used for immunoassays, immunoprecipitation, co-immunoprecipitation of protein complexes, co-immunoprecipitation of protein-nucleic acid complexes, as well as many other downstream applications. Other downstream assays could include those requiring proteins such as lectins, enzymes, or others to be coupled to the Dynabeads in stead of antibodies. Captured proteins, protein complexes and protein-nucleic acid complexes are easily separated from lysate using magnetic separation properties of Dynabeads. Magnetic separation facilitates washing, buffer changes and elution. Antibody-to-bead coupling works optimally with purified antibodies, although the coupling reaction also works well with antibodies in storage buffers that include protein additives (e.g. BSA) and/or sodium azide (NaN3). This kit is not recommended for use with antibodies that have been stabilized in glycerol
Other protein ligands (e.g. lectins, enzymes, etc.) can be covalently coupled to the surface of Dynabeads using the same beads, buffers and protocol provided in this kit.
主要试剂
Dynabeads® Antibody Coupling Kit
Magnet: e.g. DynaMag™-2
Mixer allowing rotation or tilting of tubes.
Antibodies or other proteins to be coupled
实验步骤
Day 1
1. Weigh out the appropriate amount of Dynabeads® M-270 Epoxy.
Moisture on unused beads will deactivate the reactive groups necessary for covalent antibody coupling. To avoid condensation on unused beads, make sure the beads are at room temperature prior to opening the bottle.
2. Wash the beads: Add 1 ml of C1 to the beads and mix by vortexing or pipetting.
3. Place the tube on a magnet and allow the beads to collect at the tube wall. Remove the supernatant.
4. Add the appropriate volume of antibody C1 to the washed beads and mix by vortexing or pipetting.
Example: If you are coupling 5 mg Dynabeads and your required quantity of antibodies has a volume of 100 μl, you will need to add 150 μl of C1 (i.e. 250μl C1 – 100μl Ab = 150μl.)
5. Add the appropriate volume of C2 and mix by vortexing or pipetting.
6. Incubate on a roller at 37°C overnight (16-24 hours). Make sure the fluid in the tube is mixing well. Make sure the beads do not settle. Beads settling during the overnight incubation will result in inefficient antibody coupling.
Day 2
7. Place the tube on a magnet. Allow the beads to collect at the tube wall. Remove the supernatant.
8. HB Wash: Add 0.8 or 1.6 ml (Table 2) of HB and mix by vortexing or pipetting. Place the tube on a magnet, allow the beads to collect at the tube wall, then remove the supernatant.
9. LB Wash: Add 0.8 or 1.6 ml (Table 2) of LB and mix by vortexing or pipetting. Place the tube on a magnet, allow the beads to collect at the tube wall. Remove the supernatant.
10. Short SB Wash: Add 0.8 or 1.6 ml (Table 2) of SB and mix by vortexing or pipetting. Place the tube on a magnet, allow the beads to collect at the tube wall, then remove the supernatant. Repeat the wash once more.
11. Long SB Wash: Add 0.8 or 1.6 ml (Table 2) of SB and mix by vortexing or pipetting. Incubate on a roller/rotator at RT for 15 minutes. Place the tube on a magnet, allow the beads to collect at the tube wall, then remove the supernatant.
12. Resuspend beads in the same volume of SB as was the total coupling reaction volume and store at 4°C until use. The final bead concentration is 10 mg antibody coupled beads/ml. Your beads are now covalently coupled with antibody and ready for IP, Co-IP, a her assays.
注意事项
1. Precautions
These products are designed to be used with very strong permanent magnets. People wearing a pacemaker or any other medical magnetizable implant should not use this product unless advised by a health professional; the implant could be affected or damaged by exposure to a strong magnetic field.
Keep magnetizable tools and objects out of the working area. This includes, but is not restricted to, credit cards and other products containing magnetic recording devices. Keep away from delicate instruments, watches, electronic equipment, displays and monitors. Magnets may attract steel or other magnetic material with high mechanical forces. Take care during handling. Avoid contact between two magnets. Do not pull the magnets apart if contact has been made; twist off to prevent damage to the unit or fingers.
2. Disinfection of DynaMagTM Magnets
The following materials have been tested for cleaning purposes. Spray and/or wipe the DynaMag unit with one of the following cleaning agents.
70% isopropyl alcohol
1% sodium hypochlorite solution (Bleach)
0.1N HCl solution
Other disinfectants have not been tested and may not be suitable. Do not submerge in aqueous solutions and avoid prolonged exposure to water or aqueous solutions. Clean with a damp cloth and mild detergent when exposed to harsh solvents. Do not autoclave the DynaMag magnets.
3. Storage and Stability
All kit components can be stored between 2°C and room temperature. Precautions should be taken to prevent bacterial contamination of the beads. When stored in unopened vials at 2°C – Room Temperature, the Dynabeads and buffers provided in this kit are stable until the expiration date printed on the label. Beads should not be autoclaved, but can be incubated with ethanol (70%, 1 hour) or gamma irradiated. Coated beads may be stored at 2-8°C for several weeks or even months, depending on the stability of the immobilized ligand. Coated beads should be washed once for 5 min in PBS/BSA before use. Use the magnet to collect the beads according to the washing procedure.
If a preservative is needed for storage of coated beads, a final concentration of 0.02% (w/v) sodium azide (NaN3) may be added to the storage buffer. Carefully remove before use by washing (see above). Required safety precautions must be followed when handeleing this cytotoxic material.
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