Pierce Streptavidin Magnetic Beads - Fisher Scientific
Contents
1. B Preparation of the KingFisher Instrument and Plate Set up Note The following protocol is for general use with the KingFisher Flex or KingFisher 96 Instrument Modify the protocol as needed using the Thermo Scientific BindIt Software provided with the instrument l Combine antigen sample with 10 ug of biotinylated antibody per sample Incubate 1 2 hours at room temperature or overnight at 4 C with mixing Download the SA immunoprecipitation low pH elution or SA immunoprecipitation heated elution protocol from the web site www thermo com kingfisher into the BindIt Software on an external computer Transfer the protocol to the KingFisher Flex or KingFisher 96 Instrument from an external computer See the BindIt Software User Manual for detailed instructions on importing protocols Set up the plates according to Table 2 Pierce Biotechnology PO Box 117 815 968 0747 www thermo com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 3 Table 2 Pipetting instructions for the immunoprecipitation protocol Plate Plate Name Plate Type Content Volume ee Streptavidin Beads 50 ul 1 Beads Microtiter Deep Well 96 Plate Aa Binding Wash Buffer 150 ul 2 Bead Wash Microtiter Deep Well 96 Plate Binding Wash Buffer 1 000 ul 3 Antigen Sample Microtiter Deep Well 96 Plate ai 300 ul 4 Wash 1 Microtiter Deep Well 96 Plate Binding Wash Buffer 300 ul 5 Wash 2 Microtiter Deep Well2. 96 Plate Binding Wash Buffer 300 ul 6 Wash 3 Microtiter Deep Well 96 Plate Binding Wash Buffer 300 ul Low pH Elution Elution Buffer Te E Microtiter Deep Well 96 Plate SDS PAGE Reducing 100 ul Heated Elution Sample Buffer KingFisher Flex 96 Tip 8 Tip Plate Microtiter Deep Well 96 Plate Comb for Deep Well Magnets To ensure efficient release of target antigen from the antibody replace the buffer used in Wash 3 plate 6 with 0 1 Tween 20 Detergent in water no buffering capacity Notes If using less than 96 wells fill the same wells in each plate For example if using wells Al 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 beads to Plate 1 Combine the Tip Comb with a Deep Well 96 Plate See the instrument user manual for detailed instructions The beads can be eluted into 100 ul of 0 1 M glycine pH 2 3 or 100 ul SDS PAGE reducing sample buffer If using the SDS PAGE reducing sample buffer install the KingFisher Flex or 96 Heating Block see manual for proper installation If SDS PAGE buffer is selected for elution the eluate will contain streptavidin monomers and dimers and biotinylated antibody combined with target antigen If a low pH elution buffer is selected for elution streptavidin might leach from the beads Low pH elution buffers are effective for most antibody
3. INSTRUCTIONS Thermo Pierce Streptavidin Magnetic Beads 88816 88817 2077 1 Number Description 88816 Pierce Streptavidin Magnetic Beads ml supplied at 10 mg ml in water containing 0 05 NaN 88817 Pierce Streptavidin Magnetic Beads 5 ml supplied at 10 mg ml in water containing 0 05 NaN Storage Upon receipt store at 4 C Product is shipped on an ice pack Table of Contents irode Homeen ete cere rr eret treet cer e reprercercec Tee treenerrt E E a E E E cet aeereerecprr eaters 1 Important Product InfOrmatlOn s ccsccscssasecsscessbscssocsnsusessedussscasgsuessesdsd ence agevgvsgaysend oasguneonseseoge ayowstignsqudes seedsapeecogsdebenss caueasesatsaoancusbes 2 Procedure for Manual Immunoprecipitation cccccsceesseessesscesseeecesecesecssecsaecseecseeeaeceeeseceseseaeeeeeseeseeesecaecaeecseeeaeeeeeeeeeeeeeerees 2 Procedure for Automated Immunoprecipitation cccccceescesscesecesceseceecseceaecseecaeeeseeeeeseeeeseceaeeeseceaecaeeseeeaeeeaeecseeeaeeeseeeeeeereearens 3 General Troubleshoo tri Goes se she0sscbieschcetb ach eesbetecevadidsckgabcasteodbead taadeeii deca tics EEEE SERE tates Aedes as Mila aa Teenie eis 5 Frequently Asked Questions for the Thermo Scientific KingFisher Instrument c cessssseceeeesceeeeeceseeeeeeecaeeeeseeeeeeaeeneeaeeas 5 Additional Information cccccesccssesssesseeeseeseeeecesecnecessecusecaaecaaecaeecaeecaessaecaeesaeseneseesnesenecsaecsaecaaecaeeeaesenesesesessreneeseeeeas
4. antigen interactions Executing the SA Automated Immunoprecipitation Protocol Select the protocol using the arrow keys in the instrument keypad and press Start See the KingFisher Flex or KingFisher 96 User Manual for detailed information Slide open the door of the instrument s protective cover Load the plates into the instrument according to the protocol request placing each plate in the same orientation Confirm each action by pressing Start After the samples are processed remove the plates as instructed by the instrument s display Press Start after removing each plate Press Stop after all plates are removed Pierce Biotechnology PO Box 117 815 968 0747 www thermo com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 4 General Troubleshooting Problem Possible Cause Solution Low protein recovery Proteolysis of sample Add protease inhibitors Not enough magnetic beads used for capture Increase the amount of magnetic beads used for capture Insufficient amount of target protein in the sample Increase amount of antigen sample Protein does not elute Elution conditions were too mild Increase incubation time with elution buffer or use more stringent elution buffer Multiple nonspecific bands appear in eluted sample Nonspecific protein binding to the magnetic beads Add 50 200 mM NaCl to the Binding Wash and Elution Buffers Recovered protein
5. eeaeenaes 5 Related Thermo Scientific Products cccsccssessseesseesceseeseeeseceeeeseeeccaecsaecsaecsaecaeeeseseneseeeseesseceseceaeceaecaecaecaeecaeseaeseeeeenesereerees 6 Introduction The Thermo Scientific Pierce Streptavidin Magnetic Beads provide a fast and convenient method for manual or automated immunoprecipitation protein interaction studies DNA protein pulldowns and purification of biotin labeled proteins and nucleic acids Biotinylated molecules are simply added to the streptavidin coated magnetic beads for binding A magnetic stand is used for manually removing the beads from the solution For automated processing the Thermo Scientific KingFisher Flex or KingFisher 96 Instrument is used These instruments are especially useful for large scale screening of multiple samples Pierce Streptavidin Magnetic Beads use a recombinant form of streptavidin with a mass of 53 kDa and a near neutral isoelectric point pI The protein is covalently coupled to the surface of the magnetic beads For each streptavidin molecule on the bead there are 3 biotin binding sites available Unlike avidin streptavidin has no carbohydrate groups resulting in low nonspecific binding Furthermore the magnetic beads exhibit low nonspecific binding in the presence of complex biological samples such as cell lysates The affinity between streptavidin and biotin is high requiring harsh conditions for disruption It is therefore possible to elute binding partn
6. ers in an interaction complex without co eluting the biotinylated component Table 1 Characteristics of Thermo Scientific Pierce Streptavidin Magnetic Beads Composition Streptavidin monolayer covalently coupled to magnetic bead surface Magnetization Superparamagnetic no magnetic memory Mean Diameter 1 um nominal Density 2 g cm Bead Concentration 10 mg ml Binding Capacity 55 ug biotinylated rabbit IgG mg of beads 3 500 pmol biotinylated fluorescein mg of beads Pierce Streptavidin Magnetic Beads are not supplied in RNase free solutions Pierce Biotechnology PO Box 117 815 968 0747 www thermo com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax Important Product Information Do not freeze or dry the Pierce Streptavidin Magnetic Beads Freezing or drying will cause the beads to aggregate and lose binding activity After labeling proteins or nucleic acids with biotin remove unincorporated biotin with a desalting column e g Thermo Scientific Zeba Desalt Spin Columns Product No 89882 89894 Free biotin will reduce binding capacity To minimize protein degradation include protease inhibitors e g Thermo Scientific Halt Protease Inhibitor Single Use Cocktail EDTA free Product No 78425 in the preparation of cell lysate A low pH elution may be used for single use applications To minimize streptavidin leaching do not exceed 10 minutes for the elution step in either manual or automa
7. ficient for 1 000 cm of membrane 25200 25244 Precise Protein Gels see catalog or web site for a complete listing 21955 EZ Link Micro NHS PEO Biotinylation Kit Reference 1 Chaiet I and Wolf F J 1964 The properties of streptavidin a biotin binding protein produced by Streptomycetes Arch Biochem Biophys 106 1 5 Tween is a registered trademark of ICI Americas SuperSignal Technology is protected by U S Patent 6 432 662 This product Product is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale as set forth in the Product documentation specifications and or accompanying package inserts Documentation and to be free from defects in material and workmanship Unless otherwise expressly authorized in writing Products are supplied for research use only No claim of suitability for use in applications regulated by FDA is made The warranty provided herein is valid only when used by properly trained individuals Unless otherwise stated in the Documentation this warranty is limited to one year from date of shipment when the Product is subjected to normal proper and intended usage This warranty does not extend to anyone other than the original purchaser of the Product Buyer No other warranties express or implied are granted including without limitation implied warranties of merchantability fitness for any particular purpose or no
8. is inactive Elution conditions were too stringent Use a milder elution buffer e g Thermo Scientific Gentle Elution Buffer Product No 21034 Magnetic beads aggregate Magnetic beads were frozen or centrifuged The buffer was incompatible with magnetic beads Handle the beads as directed in the instructions Frequently Asked Questions for the KingFisher Instrument Question Answer Which plates are compatible with KingFisher Flex and KingFisher 96 Instruments KingFisher 96 Plates and 96 PCR plates The KingFisher Flex Instrument is compatible with the KingFisher 24 Deep Well plates Microtiter Deep Well 96 Plates KingFisher 96 Plates and 96 PCR Plates The KingFisher 96 Instrument is compatible with the Microtiter 96 Deep Well Plates Is it possible to concentrate samples during the run Both deep well plates and KingFisher 96 Plates can be used during the same run Therefore it is possible to start the processing by using larger volumes in a deep well plate and eluting the purified sample to a smaller volume in a KingFisher 96 Plate Is it possible to heat the samples during the run The heating block is located inside the instrument and can be used automatically during the sample process All plates compatible with the KingFisher Flex Instrument can be heated using specially designed interchangeable heating blocks Why do the beads stick to the plastic tips and wel
9. ls or the eluted proteins sticks to the wells Proteins conjugated to beads and eluted proteins can nonspecifically bind to plastics Adding detergent in the Binding Wash Buffer prevents the protein conjugated to the beads from sticking 0 05 0 1 Tween 20 Detergent Also include a small amount of detergent in the Elution Buffer e g 0 05 Tween 20 Detergent or silanize the elution plate Are the reagent volumes in each well critical For best results keep the specified volumes within defined limits to avoid spillover Additional Information e Visit www thermo com pierce for additional information including the following o Tech Tip 43 Protein Stability and Storage e Visit www thermo com kingfisher for information on KingFisher Products e Inthe U S A purchase KingFisher Supplies from VWR Contact your local Thermo Fisher Scientific office to purchase KingFisher Supplies outside the U S A Pierce Biotechnology 3747 N Meridian Road PO Box 117 Rockford IL 61105 USA 5 815 968 0747 815 968 7316 fax www thermo com pierce Related Thermo Scientific Products 88800 88801 Pierce Protein A Magnetic Beads 88806 88807 Pierce Protein G Magnetic Beads 88821 88822 Pierce Glutathione Magnetic Beads 88811 88812 Pierce Magnetic TiO Phosphopeptide Enrichment Kit 24615 Imperial Protein Stain 1 L sufficient for up to 50 mini gels 34075 SuperSignal West Dura Extended Duration Substrate suf
10. n infringement Buyer s exclusive remedy for non conforming Products during the warranty period is limited to replacement of or refund for the non conforming Product s There is no obligation to replace Products as the result of i accident disaster or event of force majeure ii misuse fault or negligence of or by Buyer iii use of the Products in a manner for which they were not designed or iv improper storage and handling of the Products Current versions of product instructions are available at www thermo com pierce For a faxed copy call 800 874 3723 or contact your local distributor 2009 Thermo Fisher Scientific Inc All rights reserved Unless otherwise indicated all trademarks are property of Thermo Fisher Scientific Inc and its subsidiaries Printed in the USA Pierce Biotechnology PO Box 117 815 968 0747 www thermo com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 6
11. o ensure efficient release of target antigen from the antibody pre rinse the beads 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 96 100 C in a heating block for 5 minutes Magnetically separate the beads and save the supernatant containing 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 Note Use the Thermo Scientific Clean Blot IP Detection Reagent Product No 21230 or 21233 to prevent detection of the immunoprecipitation antibody in Western blots Procedure for Automated Immunoprecipitation A Additional Materials Required KingFisher Flex with 96 Deep Well Head Product No 5400630 or KingFisher 96 Product No 5400500 Instrument Thermo Scientific Microtiter Deep Well 96 Plate V bottom polypropylene Product No 95040450 KingFisher Flex 96 Tip Comb for Deep Well Magnets Product No 97002534 1 5 ml microcentrifuge tubes Binding Wash Buffer Tris buffered saline TBS Product No 28379 containing 0 1 Tween 20 Detergent Elution Buffer IgG Elution Buffer Product No 21004 or 21009 or 0 1 M glycine pH 2 3 Alternative Elution Buffer SDS PAGE reducing sample buffer Antigen sample Biotinylated antibody
12. ow beads to dry If necessary store beads in Binding Wash buffer before proceeding with the purification protocol Immunoprecipitation Note This protocol is a general guideline for immunoprecipitation and will require optimization for each application Combine the antigen sample with 10 ug of biotinylated antibody Incubate 1 2 hours 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 Add the antigen sample biotinylated antibody mixture to a 1 5 ml microcentrifuge tube containing pre washed magnetic beads see Section B above and incubate at room temperature for 1 hour with mixing Collect the beads with a magnetic stand and remove and save the supernatant for analysis Pierce Biotechnology PO Box 117 815 968 0747 www thermo com pierce 3747 N Meridian Road Rockford IL 61105 USA 818 968 7316 fax 2 Add 300 ul of Binding Wash Buffer to the tube and gently mix Collect the beads and then discard the supernatant Repeat this wash twice Elution Buffer Recovery of Antigen Add 100 ul of Elution Buffer to the tube Incubate the tube at room temperature with mixing for 5 minutes Magnetically separate the beads and save the supernatant containing target antigen Note If a low pH elution buffer is selected for elution streptavidin leaching might occur Low pH elution buffers are effective for most antibody antigen interactions however t
13. ted protocols Boiling the magnetic beads in SDS PAGE reducing sample buffer is acceptable for single use applications Boiling will cause bead aggregation and loss of binding activity The Pierce Streptavidin Magnetic Beads are compatible with mass spectrometry because of their low nonspecific binding Procedure for Manual Immunoprecipitation A Additional Materials Required 1 5 ml microcentrifuge tubes Binding Wash Buffer Tris buffered saline TBS Product No 28379 containing 0 1 Tween 20 Detergent Elution buffer IgG Elution Buffer Product No 21004 or 21009 or 0 1 M glycine pH 2 3 Alternate elution buffer SDS PAGE reducing sample buffer Biotinylated antibody Antigen sample Cell lysis buffer used to prepare antigen sample Magnetic stand e g Thermo Scientific MagnaBind Magnet for 6 x 1 5 ml Microcentrifuge Tubes Product No 21359 Pre washing Pierce Streptavidin Magnetic Beads Note To ensure homogeneity mix the beads thoroughly before use by repeated inversion gentle vortexing or using a rotating platform Add 50 ul 0 5 mg of Pierce Streptavidin Magnetic Beads into a 1 5 ml microcentrifuge tube Place the tube into a magnetic stand to collect the beads against the side of the tube Remove and discard the supernatant Add 1 ml of Binding Wash Buffer to the tube Invert the tube several times or vortex gently to mix Collect the beads with a magnetic stand then remove and discard the supernatant Note Do not all
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