Sera-Mag™ SpeedBeads Magnetic Protein A/G Particles
Contents
1. for detailed instructions on importing protocols 3 Set up the plates according to Table 2 Table 2 Pipetting Instructions for the Antibody Protocol Using the Microtiter Deep Well 96 Plates Plate Plate Content Volume Name ic Protein A G particles 50 ul 1 panicle Binding Wash buffer 150 ul 2 Particle wash Binding Wash buffer 1000 ul Sample 10 ul 3 Bind Binding Wash buffer 490 ul 4 Wash 1 Binding Wash buffer 500 ul 5 Wash 2 Binding Wash buffer 500 ul 6 Wash 3 Binding Wash buffer 500 ul 7 Elution Elution buffer 100 ul KingFisher Flex 96 tip comb 8 Tip plate for Deep Well magnets y Notes e If 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 To ensure bead homogeneity mix the vial thoroughly by repeated inversion gentle vortexing or rotating platform before adding the particles to plate 1 Combine the Tip Comb with a Deep Well 96 plate See KingFisher Flex or KingFisher 96 user manual for detailed instructions e Sample volume can be modified according to user preference If the sample volume is lt 500 ul dilute it to a final volume of 500 ul with binding wash buffer Executing the antibody purification protocol on the KingFisher Flex 1 Select the protocol using the arrows on the instrument key pad and press Start See KingFisher Flex User Manual for detailed information 2 Slide open the door of the instrumen2. processed remove the plates as instructed by the instrument s display Press Start after removing each plate Press Stop after all the plates are removed Troubleshooting General troubleshooting tips and suggestions for Sera Mag Speedbeads Protein A G Magnetic Particles Problem Possible Cause Solution Low amount of protein was recovered The protein degraded Insufficient magnetic particles used Insufficient target protein present in sample Add protease inhibitors Increase the amount of magnetic particles used for capture Increase amount of antigen sample Protein does not elute Elution conditions are too mild Increase incubation time with elution buffer or use more stringent elution buffer Bands at M 50 000 Elution conditions are too stringent appear on Western blot Perform elution at room temperature Multiple nonspecific bands appear in eluted sample Nonspecific protein binding to the magnetic particles Add 50 200 mM NaCl to the binding wash and or elution buffers Recovered protein was inactive Elution conditions are too stringent Use a milder elution buffer Magnetic particles aggregate Magnetic particles were frozen or centrifuged Buffer is incompatible with magnetic particles Handle the particles as directed in the instructions 6 29 1079 14AA 29 1079 14AA 7 For local office contact information visit www gelifesciences com contact www gelifesciences com ser
3. the eluted antibody To neutralize the low pH add 10 ul of neutralization buffer for each 100 ul of eluate Note 50 ul is the minimum volume of particles recommended for antibody purification Procedure for automated antibody purification 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 e KingFisher Flex 96 tip comb for Deep Well Magnets Thermo Scientific product number 97002534 e Binding wash buffer Tris buffered saline containing 0 05 Tween 20 detergent e Elution buffer 0 1 M glycine pH 2 3 e Neutralization buffer High ionic strength alkaline buffer such as a 1 M phosphate or 1 M Tris pH 7 5 9 Preparation of 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 Download the Antibody Purification protocol from the Thermo Fisher Scientific website www thermoscientific com kingfisher into the Bindlt software on an external computer 2 Transfer the protocol to the KingFisher Flex or KingFisher 96 from an external computer See Bindlt Software User Manual
4. Procedure 29 1079 14 AA Protein enrichment Sera Mag SpeedBeads Magnetic Protein A G Particles Sera Mag SpeedBeads Protein A G Magnetic Particles Table 1 provide a fast and convenient method for both manual and automated magnetic isolation of proteins using affinity binding The particles can be used for isolating antibodies from serum cell culture supernatant or ascites and for immunoprecipitation and co immunoprecipitation of antigens from cell or tissue extracts Bound antibodies or antigens are dissociated from the particles using an elution buffer The particles can be manually removed from the solution using a magnetic stand or by automation using automated magnetic particle handling systems Table 1 Characteristics of Sera Mag SpeedBeads Protein A G Magnetic Particles Composition Recombinant protein A G covalently coupled to particle surface Magnetization Superparamagnetic no magnetic memory Mean diameter 1 um nomimal Concentration 10 mg ml Binding capacity 55 85 ug IgG bound per mg of particle Particle density 2 0 g cm gelifesciences com Sera Mag SpeedBeads Protein A G Magnetic Particles contain a recombinant Protein A G M 50 500 apparent molecular weight by SDS PAGE M 40 000 to 45 000 that combines the IgG binding domains of both Protein A and Protein G Protein A G contains four Fc binding domains from Protein A and two from Protein G making it a more general and convenien
5. a mag GE Healthcare UK Limited Amersham Place Little Chalfont 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 KingFisher is a trademark of Thermo Fisher Scientific LLC All other third party trademarks are the property of their respective owners 2011 2014 General Electric Company All rights reserved Previously published Sept 2011 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 Bjorkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O 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 14AA 06 2014
6. epeated inversion gentle vortexing or using a rotating platform 1 Place 50 ul 0 50 mg of Sera Mag SpeedBeads Protein A G Magnetic Particles into a 1 5 ml microcentrifuge tube Add 150 ul of binding wash buffer to the particles and gently vortex to mix 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 gently vortex to mix for 1 min Collect particles with magnetic stand then remove and discard the supernatant 4 Dilute 10 ul of sample with 490 ul binding wash buffer Note Sample volume can be modified according to user preference If the sample volume is lt 500 ul dilute it to a final volume of 500 ul with binding wash buffer 5 Add the diluted sample to the tube containing prewashed magnetic particles and gently vortex or invert to mix 6 Incubate the samples at room temperature with mixing for 1 h 7 Collect the particles with a magnetic stand then remove and discard the supernatant 8 Add 500 ul of binding wash buffer to the tube mix well collect the particles with a magnetic stand and discard the supernatant Repeat this wash twice 9 Add 100 ul of elution buffer to the tube mix well and incubate 10 min at room temperature with occasional mixing 10 Collect the particles with a magnetic stand and then remove and save the supernatant that contains
7. her Flex or KingFisher 96 Instrument The protocol can be modified according to your needs using the Bindlt Software provided with the instrument 1 Combine antigen sample with 2 10 ug of immunoprecipitation antibody per sample Incubate 1 2 h at room temperature or overnight at 4 C with mixing 2 Enter the Immunoprecipitation protocol from Table 3 into the Bindlt Software on an external computer 3 Transfer the protocol to the KingFisher Flex or KingFisher 96 from an external computer See BindIt Software User Manual for detailed instructions on importing protocols 4 Set up plates according to Table 3 Table 3 Pipetting instructions for the immunoprecipitation protocol using the Microtiter Deep Well 96 Plates Plate Plate Content Volume Time Speed name Protein A G particles 50 ul 5s 1 Particles Binding buffer 150 ul 2 Particle wash Binding buffer 1000 ul 1min slow Antibody 500 pl 1 min slow 3 Bind Antigen sample 4 Wash 1 Binding Wash buffer 500 ul 30 s slow 5 Wash 2 Binding Wash buffer 500 ul 30 s slow 6 Wash 3 Ultrapure water 500 ul 30 s slow 7 Elution Elution buffer 100 ul 10 min slow KingFisher Flex 96 tip 8 Tipplate comb for Deep Well 10 min fast magnets Notes e If 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 gen
8. r overnight at 4 C with mixing 2 Place 25 ul 0 25 mg of Sera Mag SpeedBeads Protein A G Magnetic Particles into a 1 5 ml microcentrifuge tube 3 Add 175 ul of wash buffer to the particles and gently vortex to mix 4 Place the tube into a magnetic stand to collect the particles against the side of the tube Remove and discard the supernatant 5 Add 1 ml of wash buffer to the tube Invert the tube several times or gently vortex to mix for 1 min Collect particles with magnetic stand Remove and discard the supernatant 6 Add the antigen sample antibody mixture to a 1 5 ml microcentrifuge tube containing prewashed particles and incubate at room temperature for 1 h with mixing 4 29 1079 14AA 7 Collect the particles with a magnetic stand and then remove the flow through and save for analysis 8 Add 500 ul of wash buffer to the tube and gently mix Collect the particles and then discard the supernatant Repeat this wash twice 9 Add 500 ul of purified water to the tube and gently mix Collect the particles on a magnetic stand and discard the supernatant 10 Low pH Elution Add 100 ul of low pH elution buffer to the tube Incubate the tube at room temperature with mixing for 10 min Magnetically separate the particles and save the supernatant containing target antigen To neutralize the low pH add 10 ul of neutralization buffer for each 100 ul of eluate Alternative elution Add 100 ul of SDS PAGE reducing sample b
9. se inhibitors in preparation of cell lysates e Alow pH elution may be used for single use applications Optimal time for low pH elution is 10 min Exceeding 10 min may result in nonspecific binding and yield reduction e When using rabbit antibodies primary or secondary in downstream Western blot applications perform elution in SDS PAGE sample buffer at room temperature For all other antibody species boiling the particles in SDS PAGE sample buffer is acceptable for single use applications Boiling will cause particle aggregation and loss of binding activity e Sera Mag SpeedBeads Protein A G Magnetic Particles are compatible with small scale antibody purification and immunoprecipitation and analyses by Western blot and mass spectrometry Procedure for manual antibody purification Additional materials required e 1 5 ml microcentrifuge tubes e Sample serum concentrated cell culture supernatant or concentrated ascites e Binding Wash buffer Tris buffered saline 25 mM Tris 0 15 M NaCl pH 7 5 containing 0 05 Tween 20 detergent e Elution buffer 0 1 M glycine pH 2 3 2 29 1079 14AA e Neutralization buffer High ionic strength alkaline buffer such as a 1 M phosphate or 1 M Tris pH 7 5 9 e Magnetic stand e g MagRack 6 GE Healthcare code number 28 9489 64 Antibody purification from serum cell culture supernatant or ascites Note To ensure homogeneity mix the particles thoroughly before use by r
10. t s protective cover 3 Load the plates into the KingFisher Flex according to the protocol request placing each plate in the same orientation Confirm each action by pressing Start 4 After the samples are processed remove the plates as instructed by the instrument s display Press Start after removing each plate 5 Press Stop after all plates are removed 6 Upon completion if desired neutralize the low pH by adding 10 ul of neutralization buffer for each 100 ul of eluate 29 1079 14AA 3 Procedure for manual immunoprecipitation IP Additional materials required e 1 5 ml microcentrifuge tubes e Binding buffer Tris buffered saline 25 mM Tris 0 15 M NaCl pH 7 5 containing 0 05 Tween 20 detergent e Wash buffer 25 mM Tris 0 65 M NaCl 0 05 Tween 20 detergent pH 7 5 e Low pH elution buffer 0 1 M glycine pH 2 3 e Alternative elution buffer SDS PAGE reducing sample buffer e Antibody for immunoprecipitation e Antigen sample e Cell lysis buffer used to adjust IP reaction volume e Neutralization buffer High ionic strength alkaline buffer such as a 1 M phosphate or 1 M Tris pH 7 5 9 Immunoprecipitation Note This protocol is a general guideline for immunoprecipitation and will require optimization for each application 1 Combine the antigen sample with 10 ug of antibody Adjust the reaction volume to 500 ul with the cell lysis buffer Incubate the reaction for 1 to 2 h at room temperature o
11. t tool for investigating and purifying immunoglobulins Also Protein A G binding to immunoglobulins is not as pH dependant as Protein A Sera Mag SpeedBeads Protein A G particles are uniform colloidally stable monodispersed non porous superparamagnetic spheres made by a proprietary core shell method The core is a carboxylate modified particle made by free radical emulsion polymerization of styrene and acid monomer Two layers of magnetite Fe O are coated onto this core particle resulting in faster magnetic response times The surface is chemically modified with a proprietary method to minimize nonspecific binding of proteins Finally Protein A G is covalently bound to the particle surface The particles are supplied at 1 solids 10 mg ml in 0 05 sodium azide and are available in 1 ml 5 ml and 100 ml package sizes Important information before using Sera Mag SpeedBeads Protein A G Magnetic Particles e Do not centrifuge dry or freeze the magnetic particles Centrifuging drying or freezing will cause the particles to aggregate and lose binding activity e We recommend thoroughly mixing vortex roll or sonicate magnetic protein A G particles before use Sonication is the preferred method to resuspend the particles thoroughly and efficiently e Sonication with a probe type ultrasonicator is recommended to resuspend particles after long term storage and washing steps e To minimize protein degradation include protea
12. tle 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 29 1079 14AA 5 Executing automated immunoprecipitation protocol 1 Select the protocol using the arrows on the instrument key pad and press Start See the KingFisher Flex User or Kingfisher 96 User Manual for detailed information 2 Slide open the door of the instrument s protective cover 3 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 After the samples are
13. uffer to the tube and heat the samples at 96 C to 100 C ina heating block for 10 min Magnetically separate the particles and save the supernatant containing target antigen Note If you will be performing a Western blot using rabbit antibodies primary or secondary do not heat the samples Incubate at room temperature for 10 min with mixing Procedure for automated immunoprecipitation Additional materials required e KingFisher Flex with 96 Deep Well head Thermo Scientific product number 5400630 or KingFisher 96 Thermo Scientific product number 5400500 KingFisher Flex Microtiter Deepwell 96 plate V bottom Thermo Scientific product number 95040450 KingFisher Flex 96 tip comb for Deep Well magnets Thermo Scientific product number 97002534 e 1 5 ml microcentrifuge tubes Binding buffer Tris buffered saline containing 0 05 Tween 20 detergent e Wash buffer Tris buffered saline containing 0 05 Tween 20 detergent and 0 5 M NaCl e Low pH elution buffer 0 1 M glycine pH 2 3 Alternative elution buffer SDS PAGE reducing sample buffer Antigen sample e Cell lysis buffer used to prepare the antigen sample e Neutralization buffer High ionic strength alkaline buffer such as a 1 M phosphate or 1 M Tris pH 7 5 9 e Magnetic stand e g MagRack 6 GE Healthcare code number 28 9489 64 Instrument preparation and plate set up Note The following protocol is designed for general use with the KingFis
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