NanoLink™ Streptavidin Magnetic Beads User Manual
Contents
1. microspheres to a new 1 5 mL tube or other suitable vessel For example 5 uL 10 mg mL 50 ug is sufficient to bind 125 pmol biotinylated oligonucleotide 80 ug or a biotinylated PCR product 40 ug 500 bp Note always work with a suitable quantity of microsphere for example 50 ug in a volume of 250 uL of 1x Nucleic Acid Wash and Binding Buffer Never use less than 10 ug of beads since beads are difficult to visualize and track below this level 3 Add sufficient Nucleic Acid Binding and Wash Buffer see recipe below to bring the final volume to 0 25 mL mix gently to resuspend and wash the microspheres 4 Place the tube on a magnet for 2 min discard the supernatant 5 Remove the tube from the magnet and resuspend the washed beads in 0 25 mL of Nucleic Acid Binding and Wash Buffer 6 NanoLink streptavidin microspheres are now ready for immobilization of biotinylated PCR products or other biotinylated nucleic acids 4 2 Immobilization of Biotinylated PCR Products 1 Determine the mass of NanoLink streptavidin magnetic microspheres required for your specific application and wash as described in section 3 2 leaving the microspheres in 250 uL of Nucleic Acid Binding and Wash Buffer 2 Add a volume 5 50 uL of purified PCR product in water or 1xTi9E free of excess biotinylated primers to 0 25 mL of washed microspheres in Nucleic Acid Binding and Wash Buffer 3 Vortex gently to mix 4 Incubate for 30 mi2. tube containing the immobilized antibody on a magnetic stand for 2 min and carefully remove the supernatant 9 Wash the microsphere pellet twice using 0 25 mL 1x Antibody Binding and Wash Buffer 10 The immobilized biotinylated IgG is now ready for other downstream applications such as capture and or release of cognate antigen 4 5 Binding and Wash Buffers Microsphere Blocking Solution Nucleic Acid Binding and Wash Buffer 50 mM Tris HCl 150 mM NaCl 0 05 Tween 20 pH 8 0 1X Antibody Binding and Wash Buffer 50 mM Tris HCl 150 mM NaCl 0 05 Tween 20 pH 8 0 2X Antibody Binding and Wash Buffer 100 mM Tris HCl 300 mM NaCl 0 1 Tween 20 pH 8 0 Microsphere Blocking Solution Blocker Casein in TBS Trademark of Pierce Chemical Cat 37532 Filter the casein block solution through a 0 45 filter before using Use Hammersten grade casein for blocking streptavidin microspheres 4 6 Microsphere Blocking Procedure 1 milligram microspheres 1 10 Transfer 100 ul of NanoLink Streptavidin Magnetic Microspheres 10 mg ml to a new 1 5 ml microfuge tube Place the tube on a magnetic stand for 2 minutes carefully remove and discard the supernatant Add 1 mL of Blocker Casein in TBS filtered to the microspheres to resuspend the microspheres Place the tube on a platform shaker e g LabLine Titer TeK setting of 5 for 30 minutes to block Place the tube on a magnetic stand for 2 minutes and completely remov
3. NanoLink Streptavidin Magnetic Beads 05142010 solulink NanoLink Streptavidin Magnetic Beads User Manual Catalog M 1002 www interchim com wa interchim 211 bis Avenue Kennedy BP 1140 Agence Paris Normandie 03103 Montlugon France 33 0 1 41 32 34 40 33 0 4 70 03 88 55 Fax 33 0 1 47 91 23 90 Fax 33 0 4 70 03 82 60 e mail interchim paris interchim com e mail interchim interchim com Note This protocol can be downloaded from the appropriate category in the Solulink Library NanoLink Streptavidin Magnetic Beads User Manual Product Description Applications Particle Characteristics Using NanoLink Magnetic Beads 4 1 NanoLink Microsphere Washing Procedure 4 2 Immobilization of Biotinylated PCR Products 4 3 Dissociation of unbiotinylated PCR Strand from Immobilized PCR Product 4 4 Capture and Immobilization of Biotinylated Antibody 4 5 Binding and Wash Buffers Microsphere Blocking Solution 4 6 Microsphere Blocking Procedure 5 Examples a oat ae CON OD OD WO 10 10 11 D 1 Product Description NanoLink Streptavidin Magnetic Microspheres are nanometer sized polymer encapsulated no exposed iron super paramagnetic particles containing covalently cross linked streptavidin NanoLink streptavidin magnetic microspheres are made by covalently cross linking streptavidin to a hydrophilic surface using SoluLink s proprietary HydraLink conjugation chemistry The high surface area of
4. a known mass standard of the same size Biotin Binding Capacity The biotin binding capacity of NanoLink streptavidin magnetic particles is measured in nmol mg Biotin binding is quantitatively measured by incubating a known mass of particles 0 5 mg with a fluorescein biotin standard solution for 60 minutes and quantifying the amount of residual unbound fluorescein biotin left in solution versus negative control microspheres Size Distribution by Scanning Electron Microscopy SEM Scanning electron microscopy confirms a bimodal size distribution for NanoLink Magnetic Microspheres The streptavidin particles consist of a core polymeric particle 850 nm surrounded by a population of similar but smaller polymeric microspheres 150 nm Ad Quality control A sample of each NanoLink batch is retained for biotin binding Capacity and solids analysis The remainder is packaged refrigerated and preserved in nuclease free water containing 0 05 sodium azide to prevent microbial contamination Cleaning Surfactants are not added to this product and the particles are thoroughly washed with nuclease free water containing 0 05 sodium azide prior to packaging For some applications it may be desirable to remove residual azide using a brief wash Stability Particles should be stored at 2 8 C Do not freeze If particles are settled resuspend by suitable methods including vortexing rotary mixing or swirling NanoLink streptavidin magnet
5. e the blocking solution Wash the microspheres 4 X with 1 ml 1X Antibody Binding and Wash Buffer 50 mM Tris HCl 150 mM NaCl 0 05 Tween 20 pH 8 0 Discard the wash solution between washes After the final wash resuspend the blocked microspheres at 10 mg ml using 100 ul 1X Antibody Binding and Wash Buffer Microspheres are now ready for capture and immobilization of biotinylated antibody 5 Examples 5 1 Capture of Biotinylated Antibody Using NanoLink Streptavidin Microspheres 11 Binding of ChromaLinK Biotin Modified Bovine IgG Fixed input mass of NanoLinK 1 mg at variable input mass of biotinylated IgG T m j i Super imposed UV scans 220 420 nm of biotinylated IgG supernatants incubated with NanoLink Streptavidin Magnetic Microspheres al A r s 2 R a t io oe i n Ta 3 a te EE a ona a ON eg nE mas aaa 25 Sites i Tes n ar 6 EEFE a ias n Eaua 1 1000 ug ChromaLink Biotinylated IgG Omg Nanolink Streptavidin Microspheres Control 2 1000 ug ChromaLink Biotinylated IgG 1me Nanolink Streptavidin Microspheres 3 500ug ChromaLink Biotinylated IgG 1 mg Nanolink Streptavidin Microspheres 4 250ug ChromaLink Biotinylated IgG 1 mg Nanolink Streptavidin Microspheres 5 125 ug Chroma Link Biotinylated IgG 1 mg Nanolink Streptavidin Microspheres 6 Qug ChromaLink Biotinylated IgG 1mg Nanolink Streptavidin Microspheres Binding of ChromaLinK Biotin Modified Bovi
6. ic microspheres remain stable when stored at 2 8 C for 1 year Washing NanoLink streptavidin magnetic microspheres are washed by magnetic separation using commercially available magnetic stands Niobium magnetic stands are available in 50 mL 15 mL 1 5 mL and 96 well plate formats from various vendors NanoLink microspheres are placed on a magnetic stand for 2 3 minutes and the clarified supernatant removed without disturbing the pellet Re suspension After long term storage and settling of particles it is best to resuspend the particles thoroughly to avoid any mild _ particle to particle aggregation 4 Using NanoLink Magnetic Microspheres In order to decide how much NanoLink Streptavidin Magnetic Microspheres you will need for your specific application please refer to Table 1 gt 14nmol me gt 1300 pmol mg Biotinylated oligo 23 mer gt 2 5nmol mg NA Biotinylated gt 1 7 nmol mg 0 12 nmol mg IgG 3 biotins per IgG 250 ug meg 20 ug mg Table 1 NanoLink Streptavidin Magnetic Beads binding capacity vs other competitive bead of similar size 4 1 NanoLink Microsphere Washing Procedure 1 Resuspend NanoLink streptavidin magnetic microsphere in their original vial with the aid of a vortex mixer Mix vigorously for 1 minute to fully resuspend the microspheres Pipette up and down if necessary to fully disperse the microspheres 2 Transfer the desired volume of NanoLink streptavidin magnetic
7. n 3 3 resuspend the DNA coated NanoLink microspheres in exactly 50 uL of freshly prepared 100 mM NaOH Note Prepare daily from a 10N NaOH stock solution and molecular grade water 2 Incubate the microspheres in 100 mM NaOH at room temperature for 1 minute 3 Place the tube back on the magnetic stand for an additional 1 minute and transfer the supernatant to a new 1 5 mL tube This supernatant contains the non biotinylated DNA strand 4 Immediately neutralize the non biotinylated strand by addition of 5 2 uL 1 M glacial acetic acid Confirm the pH of the neutralized solution by spotting 1 uL on 0 14 pH paper After neutralization store the solution at 4 C for later use Note Neutralization using acetic acid requires a calibrated P 10 pipette and a known glacial acetic acid molarity 1 M to neutralize the pH If necessary after adding 5 2 ul of 1 M glacial acetic acid small incremental volumes e g 0 5 ul of either 100 mM NaOH or 1 M glacial acetic acid may need to be added to achieve neutrality Always confirm neutrality of the solution pH 7 0 using a 1 ul aliquot of the neutralized sample on colored pH paper 5 With the aid of a magnetic stand immediately wash the immobilized biotinylated nucleic acid strand on the NanoLink microspheres three times with 0 25 mL Nucleic Acid Binding and Wash Buffer 50 mM Tris HCl 150 mM NaCl 0 5 Tween 20 pH 8 Discard supernatants between washes 6 Resuspend the NanoLink mic
8. ne IgG O42 J 4 km a N per 5 ON Super imposed UV scans 220 4270 nm of biotinylated leG supernatants g CIN N mee E gG sup ot t fa feo E X A incubated with NanoLink Streptavidin Magnetic Microspheres B he sal 4 i 4 a X a t A k oa k j Gi Kea ka nal ah i K rr ee ae a t a tae gs hs a a y all Ta oo TT A A O j i 5 Ry a a a i i z k hp aur Tii T j w c ih io a b ay a jn i a D a a woa i 6 Te S a ete fe N Ta a ga tha Te a e i ea a a a Tin r aa aL T TE oo Lae Satta g at T a c a e a a a oe wy ia qi 00 wee oe etete Zero biotinylated lgG buffer blank control eae Hn T Oe EEEa LEE E a a E L AET n a a L L A A T E a E R a L L E AL A a S Ai ape 220 Fai 250 20 z0 2m 250 oan 0 yo a2 T HO 75 se a0 aii 14 ain 4ig FA Warehength mrn 1 250 ug ChromaLink Biotinylated IgG 0 mg Nanolink Streptavidin Microspheres 2 250 ug ChromaLink Biotinylated IgG 0 25 mg Nanolink Streptavidin Microspheres 3 250 ug ChromaLink Biotinylated IgG 0 50 mg Nanolink Streptavidin Microspheres 4 250 ug ChromaLink Biotinylated IgG 0 75 mg Nanolink Streptavidin Microspheres 5 250 ug Chroma Link Biotinylated IgG 1 0 mg Nanolink Streptavidin Microspheres 6 Oug ChromaLink Biotinylated IgG 1 0 mg Nanolink Streptavidin Microspheres The products offered here are for research use only Any commercial application will require a license from Soluli
9. nk The Solulink Conjugation System is patented and has multiple patents pending Please contact Solulink for information regarding licensing information Solulink products and methods may be covered by one or more of the following United States patents Nos 6 686 461 6 800 728 7 102 024 7 173 125 7 462 689 and other pending patent applications Information in this manual is subject to change without notice and does not constitute a commitment on the part of Solulink Inc It is supplied on an as is basis without any warranty of any kind either explicit or implied Information may be changed or updated in this manual at any time This document may not be copied transferred reproduced disclosed or duplicated in whole or in part without the prior written consent of Solulink Inc This documentation is proprietary information and protected by the copyright laws of the United States and international treaties The manufacturer of this documentation is Solulink Inc 12
10. nutes at room temperature preferably on a platform shaker e g Titer Tek Platform shaker Lab Line Instruments at a setting of 5 The platform shaker is used to keep the microspheres fully resuspended during the binding process Do not allow the microspheres to settle during binding for maximum capture efficiency Note For biotinylated oligonucleotides and DNA fragments smaller than 1 kb capturing for 30 minutes is suitable but for larger fragments e g 5 kb or larger binding at 40 C for 60 minutes may be required Inefficient biotinylation of the amplicon or the presence of excess free biotinylated primers will lead to reduced amplicon capture efficiency 5 After immobilization place the tube on a magnet for 2 min and carefully remove the supernatant Note Take care not to disrupt the pellet on the sides of the vessel during wash and aspiration steps For some applications the optical density of the supernatant can be used to quantify the amount of unbound DNA remaining e g 1 absorbance unit DNA 50 ug mL OD260 for double stranded DNA 6 Wash the immobilized biotinylated amplicon using 0 25 mL Nucleic Acid Binding and Wash Buffer and vortex gently to mix 7 Using a magnetic stand wash the microspheres two additional times After the final wash remove the supernatant and immediately proceed to the next section 4 3 Dissociation of Un biotinylated PCR Strand from Immobilized PCR Product 1 Immediately after step 8 Sectio
11. nylated antibodies and other proteins e Biotinylated dsDNA gDNA PCR products or biotinylated aRNA e Biotinylated oligonucleotides The main advantage of using these ultra high capacity microspheres includes reduction in the overall particle mass required to immobilize a biotinylated sample This leads to reduced costs and lower non specific background NSB Applications include separation of biotin labeled biomolecules including biotin labeled antibodies genomic DNA RNA PCR products oligonucleotides e g biotinylated oligo dT or peptides NanoLink streptavidin magnetic microspheres are also ideal for generating single stranded PCR templates by removal of the unbiotinylated competing PCR strand to dramatically increase hybridization efficiency to complementary targets NanoLink streptavidin magnetic microspheres are an affordable alternative for automated high throughput immobilization processes using 96 well magnets to affect multiplex binding and separation of nucleic acid or immunoassay biomolecules The particles are supplied as 1 solids 10 mg mL in nuclease free water with 0 05 sodium azide There are no surfactants present Particles can be washed with water prior use to remove residual azide if desired Particle Characteristics Percent Solids NanoLink streptavidin magnetic particles are packaged nominally at 1 solids 10 mg mL as measured using spectroscopic analysis set by their optical density at 600 nm versus
12. rospheres coated with the immobilized biotinylated strand in 0 25 mL Nucleic Acid Binding and Wash Buffer Leave the microspheres in this solution until used in other down stream applications 4 4 Capture and Immobilization of Biotinylated Antibody 1 Refer to Section 3 1 Table 1 to determine the mass of NanoLink Streptavidin Magnetic Microspheres required to capture and immobilize a given quantity of biotinylated lIgG Note For example 1 milligram of NanoLink Streptavidin Magnetic Microspheres will quantitatively bind 250 ug of biotinylated IgG at a biotin molar substitution ratio MSR of 3 2 Transfer the desired volume of pre blocked NanoLink Streptavidin microspheres 10 mg mL into a 1 5 mL microfuge tube Refer to Microsphere Blocking Procedure Section 3 7 3 Place the tube containing the microspheres on a magnet stand for 2 min and carefully remove the supernatant 4 Wash the microspheres once with 0 25 mL of 1x Antibody Binding and Wash Buffer 5 Place the tube containing the microspheres on a magnet stand for 2 min and carefully remove the supernatant 6 Add 0 125 mL of 2x Antibody Binding and Wash Buffer and 0 125 mL of biotinylated IgG containing sample to the microsphere pellet 7 Mix the beads well and incubate the tube on a platform shaker e g Titer Tek Platform shaker Lab Line Instruments setting of 5 at room temperature for 30 minutes to capture the biotinylated antibody 8 Place the
13. these paramagnetic microspheres when combined with SoluLink s efficient linking chemistry produces the most consistent and highest biotin binding capacity per unit mass of any streptavidin magnetic microsphere on the market Features Highest free biotin binding capacity of any microsphere gt 12 nmol mg Binds gt 2 5 nmol mg of a biotinylated oligonucleotide Binds gt 1 7 nmol mg of a biotinylated IlgG 250 ug mg 3 biotins IgG Encapsulated no exposed iron microspheres Microspheres size is less than 875 nm Fast magnetic response time 25 electromagnetic unit Paramagnetic no residual magnetism DOoOUCOUOUDUD U The particles are supplied at 1 solids 10 mg ml in nuclease free water with 0 05 sodium azide There are no surfactants present Particles can be washed prior to use to remove residual azide if desired but is not necessary for most applications Covalently cross linked streptavidin Outer core hydrophilic polymer surface aa Go x g seg Iron magnetite central layer si D jae oleae Taf 2 Applications 3 NanoLink streptavidin magnetic microspheres possess the highest biotin binding capacity of any commercially available polymer encapsulated streptavidin particle These microspheres are particularly suited for high throughput robotic applications where high biotin loads must be immobilized and separated using a suitably strong magnet The microspheres can be used to immobilize e Bioti
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