Sera-Mag™ SpeedBeads Streptavidin
Contents
1. the presence of complex biological samples The affinity between streptavidin and biotin is very high requiring harsh conditions for disruption such as SDS PAGE reducing sample buffer Therefore it is possible to elute binding partners in an interaction complex without co eluting the biotinylated component Sera Mag SpeedBeads combine the advantages of a high surface area high affinity and high specific activity They are colloidally stable in the absence of a magnetic field However the particles can be separated rapidly and completely from suspension when a magnetic field is applied Binding of biotinylated ligands to streptavidin groups on the surface is easily accomplished using standard avidin biotin technology In the past achieving high activity and stable binding of solid phase ligands has been a major difficulty Compounds that are difficult to attach to microparticle surfaces by conventional means may be amenable to biotinylation Due to the high affinity of the avidin biotin reaction binding biotinylated compounds to Sera Mag SpeedBeads may improve specific activity In such cases biotinylation may be carried out in aqueous or organic solvent Then the biotin derivative can be bound to Sera Mag SpeedBeads simply by mixing in appropriate buffer conditions For example nucleic acids which adsorb poorly to microparticle surfaces are readily bound to Sera Mag SpeedBeads particles after biotinylation The use of magnetic partic2. Buckinghamshire HP7 9NA UK GE and GE monogram are trademarks of General Electric Company Sera Mag is a trademark of General Electric Company or one of its subsidiaries Tween is a trademark of the Croda Group of Companies All other third party trademarks are the property of their respective owners O 2011 2014 General Electric Company All rights reserved Previously published June 2010 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29 1079 15AA 06 2014
3. Procedure 29 1079 15 AA Protein enrichment Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles Table 1 use a non surfactant non protein blocking reagent and provide low nonspecific binding NSB and high binding capacity for biotinylated target molecules Typical applications include e improvement and simplification of ligand binding e affinity purifications e immunoprecipitation e protein interaction studies e DNA protein pulldowns e purification of biotin labeled proteins and nucleic acids e other molecular biology applications Biotinylated molecules are bound to the magnetic particles which are removed from the solution using a magnetic field For manual processing a simple magnetic stand can be used An automated platform can also be used for magnetic purification Streptavidin is a M 60 000 protein from Streptomycetes avidinii 1 The protein is a tetramer containing four biotin binding sites and is covalently coupled to the surface of blocked magnetic particles There are two to three biotin gelifesciences com binding sites available for each streptavidin molecule bound to the microparticle surface Unlike avidin streptavidin has a low isoelectric point pl 5 and no carbohydrate groups resulting in low non specific binding Furthermore the protein is coupled to magnetic particles that exhibit very low non specific binding in
4. anual for detailed instructions on importing protocols 29 1079 15AA 3 4 Set up the plates according to Table 1 which will subsequently be placed into the KingFisher Flex or KingFisher 96 instrument Notes e f using less than 96 wells fill the same wells in each plate For example if using wells A1 through A12 use these same wells in all plates e To ensure particle homogeneity mix the vial thoroughly by repeated inversion gentle vortexing or rotating platform before adding the particles to plate 1 e Combine the Tip Comb with a Deep Well 96 plate See KingFisher Flex or KingFisher 96 user manual for detailed instructions e The particles can be eluted into 100 ul of 0 1 M glycine pH 2 3 or 100 ul SDS PAGE reducing sample buffer If using SDS PAGE reducing sample buffer in a heated elution install the KingFisher Flex or 96 Heating Block see manual for proper installation to heat samples at 96 C to 100 C for 10 min e If you select SDS PAGE reducing sample buffer for elution and will be performing a Western blot using rabbit antibodies primary or secondary do not heat the samples Incubate at room temperature for 10 min e If low pH elution buffer is selected for elution neutralize the pH using 10 ul neutralization buffer for each 100 ul of eluate upon run completion e To limit evaporation select Mix and Slow speed under the subheading Heating Action Executing the SA Immunoprecipitation P
5. ecipitation with biotinylated antibody Additional Materials Required e KingFisher Flex with 96 deep well head Thermo Scientific product number 5400630 or KingFisher 96 Thermo Scientific product number 5400500 e KingFisher Flex Microtiter Deepwell 96 plate V bottom Thermo Scientific product number 95040450 50 pcs e KingFisher 96 tip comb for Deep Well magnets Thermo Scientific product number 97002534 10 x 10 pcs box e 1 5 ml microcentrifuge tubes e Sera Mag Buffer Kit code number 281111 Hydridization wash and elution buffers included in kit e Alternate elution buffer SDS PAGE reducing sample buffer e Antigen sample e Biotinylated antibody Preparation of KingFisher instrument and plate set up Note The following protocol is designed for general use with the KingFisher Flex or KingFisher 96 Instrument The protocol can be modified according to customer needs using the Thermo Scientific BindIt software provided with the instrument 1 Combine antigen sample with 10 ug of biotinylated antibody per sample Incubate 1 to 2 h at room temperature or overnight at 4 C with mixing 2 Download the SA Immunoprecipitation low pH elution or SA Immunoprecipitation heated elution protocol from www thermoscientific com kingfisher into the Bindlt software on an external computer 3 Transfer the protocol to the KingFisher Flex or KingFisher 96 from an external computer See the BindIt software user m
6. les as a solid phase support in immunoassays and molecular biology applications is well documented Standard protocols are available to biotinylate a wide range of ligands including proteins nucleic acids haptens peptides and other molecules Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles are uniform colloidally stable monodispersed non porous super paramagnetic spheres made by a proprietary core shell process The core is a carboxylate modified particle made by free radical emulsion polymerization of styrene and acid monomer Magnetite Fe O is coated onto this core particle and then encapsulated with propriety polymers Finally the surface is blocked with a proprietary method to help prevent the nonspecific binding of proteins Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles are nominal 1 um particles with highly active streptavidin covalently bound to the surface They are supplied at approximately 1 solids 10 mg ml in 0 05 sodium azide Table 1 Characteristics of Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles Streptavidin monolayer covalently coupled Composition to bead surface Magnetization Superparamagnetic no magnetic memory Mean diameter 1 um nomimal Bead concentration 10 mg ml bead weight volume 1 solids Binding capacity per mg of bead 3500 pmol biotinylated fluorescein Particle density 2 0 g cm Note Sera Mag SpeedBeads Streptavidi
7. n Blocked Magnetic Particles are not supplied in RNase free solutions Important information before using Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles e Do not freeze or dry Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles This causes the particles to aggregate and lose binding activity e After labeling proteins or nucleic acids with biotin remove unincorporated biotin with a desalting column Free biotin will reduce the binding capacity of the particles 2 29 1079 15AA e To minimize protein degradation include protease inhibitors in the preparation of cell lysate e Alow pH elution may be used for single use applications To limit leaching of streptavidin do not exceed 10 min for the elution step in either manual or automated protocols e Boiling the magnetic particles in SDS PAGE reducing sample buffer is acceptable for single use applications Boiling causes microparticle aggregation and loss of binding activity e Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles can be used successfully with mass spectrometry because the non specific binding is very low Procedure for manual immuno precipitation using a biotinylated antibody Additional materials required e Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles e 1 5 ml microcentrifuge tubes e Sera Mag Buffer Kit code number 281111 Hydridization wash and elution buffers included in kit e Alternate elution b
8. ral troubleshooting tips and suggestions for Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles Problem Possible Cause Solution Low protein recovery Protein does not elute Multiple non specific bands appear in eluted sample Proteolysis of sample Not enough magnetic beads used for capture Insufficient target protein present in sample Free biotin present in sample Elution conditions are too mild Non specific protein binding to the magnetic beads Magnetic beads were frozen or centrifuged Add protease inhibitors Increase the amount of magnetic beads used for capture Increase amount of antigen sample Dialyze biotinylated antibody or pass it through a desalting column prior to binding to the magnetic beads Increase incubation time with elution buffer or use more stringent elution buffer Add 50 200 mM NaCl to the binding wash and or elution buffers Handle the beads as Magnetic beads directed in the aggregate Buffer used is instructions incompatible with magnetic beads References 1 Chaiet and Wolf F J The properties of streptavidin a biotin binding protein produced by Streptomucetes Arch Biochem Biophys 106 1 5 1964 29 1079 15AA 5 6 29 1079 15AA 29 1079 15AA J For local office contact information visit www gelifesciences com contact www gelifesciences com sera mag GE Healthcare UK Limited Amersham Place Little Chalfont
9. re to a 1 5 ml microcentrifuge tube containing prewashed magnetic beads see above and incubate at room temperature for 1 h with mixing 6 Collect the particles with a magnetic stand remove the supernatant and save for analysis 7 Add 300 ul of binding wash buffer to the tube and gently mix Collect the particles and then discard the supernatant Repeat twice 8 Elution buffer recovery of antigen Add 100 ul of elution buffer to the tube Incubate the tube at room temperature with mixing for 5 min Magnetically separate the particles and save the supernatant containing target antigen Note If a low pH elution buffer is selected for elution streptavidin may leach from the particles Low pH elution buffers are effective for most antibody antigen interactions However to ensure efficient release of target antigen from the antibody prerinse the particles with 300 ul 0 1 Tween 20 in water no buffering capacity before adding low pH elution buffer Alternate Elution SDS PAGE reducing sample buffer recovery of antigen Add 100 ul of SDS PAGE reducing sample buffer to the tube and heat the samples at 96 C to 100 C in a heating block for 5 min Magnetically separate the particles and save the supernatant containing the target antigen Note If SDS PAGE buffer is selected for elution the eluate will contain streptavidin monomers and dimers and biotinylated antibody along with target antigen Procedure for automated immunopr
10. rotocol on the KingFisher 96 1 Select the protocol using the arrow keys in the instrument keypad and press Start See the instrument user manual for detailed information 2 Slide open the door of the protective cover of the instrument Load the plates into the instrument according to the protocol request placing each plate in the same orientation Confirm each action by pressing Start 4 29 1079 15AA 3 After sample processing remove the plates as instructed by the instrument display Press Start after removing each plate 4 Press Stop after all plates are removed Table 1 Pipeting instructions for immunoprecipitation protocol Plate Plate Plate type Content Name 1 Beads Microtiter Deep Streptavidin 50 ul Well 96 Plate beads 2 Bead wash Microtiter Deep Binding Wash 150 ul Well 96 Plate buffer 3 Antigen sample Microtiter Deep Binding Wash 1000 ul Well 96 Plate buffer 4 Wash 1 Microtiter Deep Antibody 300 ul Well 96 Plate Antigen sample 5 Wash 2 Microtiter Deep Binding Wash 300 ul Well 96 Plate buffer 6 Wash 3 Microtiter Deep 0 1 Tween 300 ul Well 96 Plate 20 in water 7 Elution Immuno Microtiter Deep Elution buffer 100 ul precipitation Well 96 Plate low pH elution 7 Elution Microtiter Deep SDS PAGE 100 ul Immuno Well 96 Plate reducing sample precipitation buffer heated elution 8 Tip plate Microtiter Deep King Fisher Flex Well 96 Plate 96 tip comb for Deep Well magnets Gene
11. uffer SDS PAGE reducing sample buffer e Biotinylated antibody e Antigen sample e Cell lysis buffer used to prepare antigen sample e Magnetic stand for 1 5 ml tube e g MagRack 6 GE Healthcare code number 28 9489 64 Prewashing Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles Note To ensure homogeneity mix the particles thoroughly before use by repeated inversion gentle vortexing or using a rotating platform 1 Aliquot 50 ul 0 5 mg of Sera Mag SpeedBeads Streptavidin Blocked Magnetic Particles into a 1 5 ml microcentrifuge tube 2 Place the tube into a magnetic stand to collect the particles against the side of the tube Remove and discard the supernatant 3 Add 1 ml of binding wash buffer to the tube Invert the tube several times or vortex gently to mix Collect the particles with a magnetic stand then remove and discard the supernatant Note Do not allow the particles to dry If necessary store them in binding wash buffer prior to proceeding with purification protocol Immunoprecipitation Note This protocol is a general guideline for immunoprecipitation and requires optimization for each application 4 Combine antigen sample with 10 ug of biotinylated antibody Incubate 1 to 2 h at room temperature or overnight at 4 C with mixing Note Dilute each sample to a minimum volume of 300 ul with cell lysis buffer or binding wash buffer 5 Add the antigen sample biotinylated antibody mixtu
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