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1. Rad Bulletin 5821 Shezifi D et al 2012 Ligand Immobilization in Protein Interaction Studies An Unattended Amine Coupling Protocol with Automatic Co Injection Activation BioRadiations May 2012 21 NeutrAvidin is a trademark of Thermo Fisher Scientific Inc Tween is a trademark of ICI Americas Inc For technical support call your local Bio Rad office or in the U S call 1 800 424 6723 This product is for research use only 2012 Bio Rad Laboratories Inc Reproduction in any form either print or electronic is prohibited without written permission of Bio Rad Laboratories Inc Life Science Group Bulletin 6295 Rev A US EG Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 952. Reagent Orientation Volume pl Flow Rate pl min 1 1x EDAC sulfo NHS Vertical 150 30 2 4 6 mM alkylamine Vertical 150 30 3 1 M ethanolamine HCI Vertical 150 30 Note 1x EDAC sulfo NHS contains a 1 1 mixture of EDAC and sulfo NHS These components are included with the ProteOn amine coupling kit We recommend following the instructions shipped with the kit to prepare the activation reagents for this protocol The final concentrations of the activation reagents are 20 mM EDAC and 5 mM sulfo NHS Partial Surface Modification The extent of surface modification can be used to control the liposome capturing level and configuration Lower surface modification will reduce the capturing capacity and in many cases improve the resistance to nonspecific binding Lower modification will also increase the chances of capturing intact liposomes while higher modification will increase the tendency of liposomes to deform and even open into lipid bilayers spread over the surface Partial surface modification refers to varying the immobilization level of undecylamine by controlling the immobilization conditions This can be achieved by controlling the activation level undecylamine concentration or injection volume Otherwise mixing undecylamine with ethanolamine HCI the deactivation solution from the amine coupling kit is an easy to use and efficient alternative For fine tuned control of the surface modification level we recommend varying the undecylami
3. activation level because it can lead to overactivation and subsequent multivalent linking of the ligand It is possible to eliminate the manual mixing of the activation reagents by using the Co inject function of the ProteOn system Refer to the article Ligand Immobilization in Protein Interaction Studies An Unattended Amine Coupling Protocol with Automatic Co Injection Activation in the May 2012 online issue of BioRadiations Note 1x EDAC sulfo NHS contains a 1 1 mixture of EDAC and sulfo NHS components that are included with the ProteOn amine coupling kit We recommend following the instructions shipped with the kit to prepare the activation reagents The final concentrations of the activation reagents are 20 mM EDAC and 5 mM sulfo NHS Ligand Immobilization We recommend preparing the ligand in a concentration range of 0 5 pg ml to 25 ug ml Typically using a slow flow rate of 30 ul min is suggested to reduce ligand use during ligand injection the contact time may vary from 1 min to over 10 min depending on the immobilization level needed The ligand coupling buffer is 1 pH unit lower than the ligand pl to facilitate charge attraction between the positive ligand and the negative chip surface Low ionic strength is also required to enhance charge attraction These conditions provide a starting point for further optimizing experimental conditions To achieve the desired immobilization level choose from the two following approa
4. and Liposome Capturing Kit Refer to the product insert of the liposome capturing kit for details on how to use this kit part number 10024332 Activation Conditioning and Liposome Capture Inject the biotin ssDNA solution for surface activation Then precondition the chip surface with the lipid modification conditioning solution 20 mM CHAPS before liposome capture Next inject the chol dsDNA 1 tagged liposome solution If an additional liposome layer is needed inject the chol dsDNA 2 solution 0 4 UM and allow the signal to stabilize for 5 min then inject the chol dsDNA 1 tagged liposome solution Repeat this step to form multiple liposome layers Table 3 LCP chip activation conditioning and liposome capture parameters Injection Reagent Orientation Volume pl Flow Rate pl min 1 1 3 UM biotin ssDNA Vertical 50 30 2 20 mM CHAPS Vertical 150 30 3 1 mg ml chol dsDNA 1 Vertical 150 30 tagged liposomes 4 0 4 uM chol dsDNA 2 Vertical 50 30 5 1 mg ml chol dsDNA 1 Vertical 150 30 tagged liposomes Note Injections 4 and 5 are optional GLC Lipid Kit Refer to the product insert of the GLC lipid kit for details on how to use this kit part number 10023826 Surface Modification Use the activation and deactivation reagents from the ProteOn amine coupling kit Inject 1x EDAC sulfo NHS lipid modification solution and 1 M ethanolamine HCI sequentially Table 4 GLC surface modification parameters Injection
5. are applied When using the HTG HTE sensor chips non electrostatic NSB can also be caused by proteins with a sequence containing a few adjacent histidine residues which have a low affinity for the nickel II activated tris NTA surface Molecules that can potentially exhibit electrostatic and non electrostatic NSB are listed in Table 6 Analyte Non Ligand Non Ligand electrostatic i analyte electrostatic analyte NSB interaction Analyte NSB interaction Ligand b b gt Ligand b d Surface layer Surface layer Gold substrate Gold substrate Fig 5 Non electrostatic NSB on the chip surface Table 6 Molecules with a propensity for NSB Molecules in Samples Type of NSB Positively charged proteins with a Electrostatic NSB pl gt pH of the running buffer Relatively small molecules containing thiol groups or related forms of sulfur Non electrostatic NSB such as cysteine containing peptides known to show high affinity to gold Molecules that naturally bind polysaccharides such as lectins may exhibit NSB to the Non electrostatic NSB alginate containing binding layer Biological solutions such as serum Electrostatic NSB and crude lysates or supernatants non electrostatic NSB Techniques for Reducing or Eliminating NSB on All Sensor Chips Suggested changes in experiment design to reduce NSB 1 Optimize the appropriate running buffer and sample buffer for the application 2 Purify th
6. histidine tag capturing chips or HTX chips we recommend preparing the ligand in a concentration range of 0 5 ug ml to 25 ug ml and using a slow flow rate of 30 ul min for the ligand injection to reduce ligand use The contact time may vary from 1 min to over 10 min depending on the immobilization level needed These conditions provide a starting point for the further optimization of experimental conditions Perform ligand injection immediately after the activation step to avoid nickel II ion leakage and consequently a reduced immobilization level Table 2 HTX chip activation and ligand capture parameters Injection Reagent Orientation Volume pl Flow Rate pl min il 10 mM NiSO Vertical 60 30 2 0 5 25 ug ml ligand Vertical Flexible 30 The ProteOn HTG and HTE sensor chips are designed to capture histidine tagged proteins directly from crude media and purified proteins When capturing ligand from crude samples dilute the ligand sample before the capture to reduce nonspecific binding We recommend performing a significant dilution for example by 100 fold depending on the amount of active ligand in the sample Refer to bulletin 6802 for more detailed techniques to reduce nonspecific binding Note The HTG and HTE chips require activation but not deactivation Ligand injection should be performed immediately after the activation step to avoid nickel Il ion leakage and consequently a reduced binding level LCP Sensor Chip
7. vial caps and microplate sealing films to prevent vacuum formation inside the container during aspiration 2 Degas the sample and reagent solutions 3 Verify that the volume of sample and reagent solutions is sufficient before injection We Regeneration Regeneration removes the analyte or ligand analyte complex on the chip surface to prepare the surface for the next experiment without damaging the ligand and or chip surface Regeneration conditions should be optimized for each interaction The reproducibility of repeated analyte injections is typically used to check the performance of regeneration With a good regeneration protocol the sensorgrams of repeated analyte injections should overlap when viewed in the same window GLX and NLC Sensor Chips For GLX and NLC chips the ligand is bound to the chip surface by covalent or very high affinity noncovalent linking Regeneration is used to remove the analyte while keeping the ligand active on the chip surface The regeneration conditions should be optimized to a balance that is strong enough to completely remove the analyte but not so harsh as to damage the ligand Some recommended conditions for different interaction systems are listed below Table 8 GLX and NLC chip regeneration reagents Ligand Analyte Recommended Reagent Protein antibody Protein peptide 10 mM glycine pH 1 5 3 0 1 phosphoric acid Peptide nucleic acid Protein peptide 0 01 0 5 SDS 5 10 mM NaOH Nucle
8. 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000 12 1094 1012 Sig 1211
9. Flow Rate l min l 0 5 SDS Horizontal 30 30 2 50 mM NaOH Horizontal 30 30 3 100 mM HCI Horizontal 30 30 4 300 mM EDTA Horizontal 30 30 5 0 5 SDS Vertical 30 30 6 50 mM NaOH Vertical 30 30 7 100 mM HCI Vertical 30 30 8 300 mM EDTA Vertical 30 30 Note 1 In conditioning we recommend using the same buffer used for running experiments When working with buffers containing metal ions that form hydroxide precipitates however eliminate the NaOH injections 2 Trehalose is used as a protective layer for the dry NeutrAvidin on the NLC chip surface It will be completely removed by continuous buffer flow over the chip surface However conditioning is highly recommended to ensure complete removal of the protective layer 3 For the LCP chip used with the ProteOn liposome capturing kit perform conditioning after biotin ssDNA capture and before liposome capture For the GLC chip used with the ProteOn GLC lipid kit perform conditioning after surface modification and before liposome capture Refer to the Ligand Immobilization section for more details Ligand Immobilization Ligand immobilization refers to the attachment of a ligand to the chip surface either by irreversible covalent bonding or by reversible capturing using a capture agent Refer to bulletin 5821 for details on achieving optimal ligand immobilization Desired Ligand Immobilization Level The immobilization level R or amount of ligand immobilized on the chip surface shou
10. ProteOn Sensor Chips ProteOn Sensor Chips Tips and Techniques Contents ProteOn Sensor Chips Chips mobilization Desired Ligand Immobilization Level Optimizing Immobilization Conditions GLX Sensor Chips Activation Ligand Immobilization Deactivation NLC Sensor Chip Ligand Capture HTX Sensor Chips Activation and Ligand Capture LCP Sensor Chip and Liposome Capturing Kit Activation Conditioning and Liposome Capture GLC Lipid Kit Surface Modification Partial Surface Modification Liposome Capture Troubleshooting Ligand Immobilization Amine Coupling with GLX Chips Typical Ligand Buffer Conditions Activation Quality Ligand Activity Ligand Capture with the NLC Chip Stabilization Analyte Injection Troubleshooting Analyte Injection Nonspecific Binding Electrostatic NSB Non Electrostatic NSB Techniques for Reducing or Eliminating NSB on All Sensor Chips Bulk Effect Correcting for Bulk Effect Bubble Formation Tips for Reducing Bubble Formation Regeneration GLX and NLC Sensor Chips HTX Sensor Chips LCP Chip and Liposome Capturing Kit GLC Lipid Kit Related Literature OO WMAWDWMDOANNNWANNOOOGGaaAHR A owon ProteOn Sensor Chips All ProteOn sensor chips are designed for use with the ProteOn XPR36 protein interaction array system Each sensor chip is a gold coated glass prism containing surface chemistry used for the immobilization of biomolecules ligands to the chip surface This enables interactions with oth
11. ate fitting to the binding model In practice however it is very difficult to determine whether NSB is similar on the interaction and reference surfaces There are cases where the ligand molecules on the interaction surface block NSB on the chip surface This leads to a higher NSB response on the reference surface and results in incorrectly referenced data or even negative responses At present NSB is one of the most difficult factors to optimize in label free biomolecular interaction analysis There are two main strategies used in SPR biosensors to overcome NSB 1 Using a reference surface that is as similar to the ligand surface as possible Optimally a reference protein unrelated to the ligand should be bound at the same density as the ligand 2 Minimizing NSB by optimizing the buffer conditions and surface chemistry 13 A No NSB i Analyte on ligand channel ii Analyte on reference channel 2007 1 2007 5 T 1604 f 1605 2 1207 a 20 fe Qa 8 2 i aS EEE ee T T T T T T T T 40 0 40 80 120 160 200 240 B NSB i Analyte on ligand channel ii Analyte on reference channel 5 6007 A 6004 z z g 4004 j 4004 S P 2004 200 f o at a 01 T T T T T T 01 T T T T 100 0 100 200 300 400 100 0 100 200 300 400 Time sec Time sec Fig 3 Comparison of responses on reference surfaces showing only bulk effect a refractive in
12. buffer Correcting for Bulk Effect 1 Apply proper referencing 2 Match the refractive index of the sample buffer and running buffer 3 In experiments where analytes are dissolved in a cosolvent with a high refractive index such as DMSO the reference surface produces a larger bulk solvent response than the ligand surface because of the larger concentration of cosolvent near the reference surface This effect is due to the exclusion of cosolvent by the ligand near the ligand surface The resulting difference in bulk effect causes inaccurate reference subtraction To correct for the difference in bulk effect between interaction and reference surfaces excluded volume correction is applied refer to bulletin 5822 for more details Bubble Formation Separation air bubbles are intentionally created between the sample and running buffer to prevent mixing during sample aspiration No bubbles should be injected into the ligand or analyte channels Spikes in the sensorgram usually indicate the injection of bubbles Small spikes can be completely removed in sensorgram processing but large spikes may cause inaccuracy in experimental results Problems with bubbles during the injection step 1 Bubbles at the beginning of an injection are usually coupled with an injection delay in one or more channels 2 Responses may vary in intensity among different channels injections and times Techniques for Reducing Bubble Formation 1 Use prepierced
13. ches 1 Set two consecutive ligand injection steps with a pause step in between The first ligand injection step is short and measures the ligand immobilization rate You may fine tune the second ligand injection according to the ligand immobilization rate at the pause step to achieve the desired immobilization level 2 Set a single long ligand injection step and monitor the ligand immobilization process When the desired immobilization level is reached hit the Abort button to end the ligand injection step Deactivation Deactivation uses 1 M ethanolamine HCl at pH 8 5 to block any remaining activated carboxyl group on the chip surface It is performed in the vertical direction the same direction as the activation and ligand immobilization injections NLC Sensor Chip Ligand Capture We recommend preparing the ligand in a concentration range of 0 5 pg ml to 25 pg ml Typically using a slow flow rate of 30 l min is suggested to reduce ligand use during ligand injection the contact time may vary from 1 min to over 10 min depending on the immobilization level needed These conditions provide a starting point for further optimization of experimental conditions Note The NLC chip does not need activation and deactivation with biotin is optional HTX Sensor Chips Activation and Ligand Capture Refer to the product insert included with the HTG and HTE reagent kit for details on how to use this kit part number 10021524 When using the
14. cupy the available binding sites on the chip surface and result in low binding levels typically of a few tens of RU 3 If the ligand is biotinylated and captured on the chip surface but does not show a binding response with the analyte over biotinylation may have occurred To avoid this we recommend preparing the ligand in a stoichiometry of 1 1 one biotin molecule per ligand molecule This also prevents cross linking of the ligand Alternatively carry out the biotinylation reaction in a low pH buffer 50 mM acetate at pH 5 5 to favor the selective biotinylation of alpha amino groups which leaves the lysine residues unblocked Stabilization Stabilization is the step between ligand immobilization and analyte injection Injecting running buffer or regeneration solutions removes any noncovalently bound ligand molecules from the chip surface Thus stabilization creates the stable baseline required to perform the interaction analysis Stabilization involves performing one or more injections of either the running buffer or regeneration solutions that do not affect the immobilized ligand Allowing the baseline to stabilize for 30 min is recommended to obtain high quality kinetic analysis For the NLC chip we recommend injecting 1 M NaCl in the stabilization step Analyte Injection In the ProteOn system ligands and analytes are typically injected at perpendicular directions in the 6 x 6 configuration to perform the interaction analysi
15. d immobilization conditions correct the conditions 2 If low ligand activity is caused by steric hindrance resulting from ligand orientation there are two potential solutions the capture surface and protected immobilization methods Figure 2 A Capture surface method Capture reagent Ww Ww SN Surface Surface Analyte Q Q 9 Ligand Y Y Y Ligand f f N gt Capture reagent Y Ww Y gt Capture reagent Y Y Y as oe es Surface Surface B Protected immobilization method Protected ligand EE mE mE mm VA YY ONG iiscs A oF ee a a Analyte Q Q gt gt Ligand 4 NA ON Ligand a a ae i i a S Surface Surface Fig 2 The capture surface method A and protected immobilization method B for eliminating steric hindrance resulting from ligand molecule orientation 3 If the ligand is vulnerable when using amine coupling use a capture surface chemistry to reversibly capture the ligand such as a chip surface functionalized with an antibody or NeutrAvidin Note that the ligand should be biotinylated or histidine tagged to use the biotin or histidine tag binding surface chemistry Ligand Capture with the NLC Chip Tips for capturing ligands with NLC chips 1 It is important to assess the result of biotinylation after ligand preparation If the biotinylation step was not successful ligand capture will not be observed on NLC chips 2 If excess biotin is not removed properly from the ligand sample it will oc
16. dex difference between the sample buffer and running buffer A or exhibiting NSB B A ii for the analyte injected in the reference channel showing no NSB the analyte response is flat during the injection and returns to zero at the end of the injection B ii for the analyte injected in the reference channel showing NSB the analyte response exhibits curvature during the injection and does not return to zero at the end of the injection Electrostatic NSB NSB is most commonly caused by the electrostatic attraction of a positively charged analyte or other sample components to the negatively charged surface layer of the sensor chip Figure 4 This type of NSB termed electrostatic NSB is common when the analyte is a protein with a pl higher than the pH of the running buffer Analyte 7 i O O Electrostatic Ligand Electrostatic Ligand NSB _ analyte NSE i _ analyte Analyte interaction interaction oO Ligand bd bd Ligand bd bd 0 Surface layer Surface layer Gold substrate Surface layer Fig 4 Electrostatic NSB on the chip surface 14 Non Electrostatic NSB Sources of NSB also include chemical interactions of the analyte or other components with the binding layer such as hydrophobic interactions hydrogen bonding or binding to nanoscopic areas of exposed gold on the surface Figure 5 This type of NSB termed non electrostatic NSB is usually observed when sticky or crude analyte samples
17. e analyte or sample when possible 3 Try different immobilization surface chemistries for example use a biotinylated ligand 4 Swap the ligand and the analyte if only the analyte shows NSB 5 Retest your binding assay with a fresh chip preferably from a different lot to eliminate chip specific defects 15 Table 7 Techniques for reducing or eliminating NSB on all sensor chips Methods Details Non Electrostatic Electrostatic NSB NSB Increase the Increase the salt concentration buffer salt to up to 500 mM in the analyte concentration buffer and running buffer using e NaCl for example to shield the electrostatic charges It is important to verify that the high salt concentration does not affect the ligand or analyte activity Increase the Increase the pH of the analyte buffer pH buffer and running buffer to reduce the positive charges contributing to electrostatic NSB It is important to verify that the high pH does not affect the ligand or analyte activity Add 0 05 Add 0 05 Tween 20 and or Tween 20 and 0 1 BSA to the running buffer or 0 1 BSA to reduce both electrostatic and non electrostatic NSB 0 1 BSA may also be used to saturate the chip surface to block potential NSB sites Create an Create an appropriate reference appropriate surface by capturing a reference reference protein unrelated to the ligand surface such as BSA to the same level as the ligand The reference protein b does no
18. er biomolecules analytes to create a measurable SPR response used in kinetic analysis and other applications Refer to bulletin 5404 for details on the surface chemistry of each chip For more information about the ProteOn instrument and instructions for running experiments see the ProteOn XPR36 protein interaction array system user manual part number 10021411 ProteOn Sensor Chips The ProteOn family of chips features outstanding performance in kinetic analysis high binding capacities high sensitivity for the detection of low molecular weight analytes uniform spot to spot response minimal baseline drift bar codes and long term storage stability Each ProteOn sensor chip is suitable for particular applications including the following ProteOn GLC sensor chip for protein protein interaction analysis ProteOn GLM sensor chip for protein small molecule and protein protein interaction analysis ProteOn GLH sensor chip for protein small molecule interaction analysis ProteOn NLC sensor chip for DNA protein and protein protein interaction analysis ProteOn HTG sensor chip for protein protein and protein peptide interaction analysis ProteOn HTE sensor chip for protein small molecule interaction analysis ProteOn LCP sensor chip for capturing lipid assemblies for lipid protein lipid small molecule and membrane protein protein interaction analysis The sensor chip cartridge labe
19. he chip surface Table 11 GLC chip regeneration conditions Injection Reagent Orientation Volume pl Flow Rate pl min 1 20 mM CHAPS Vertical 150 30 19 Ordering Information Catalog Sensor Chips 176 5011 176 5012 176 5013 176 5021 176 5031 176 5033 176 5041 Sensor Chip Application Kits 176 2300 176 2350 176 2500 176 2600 Reagent Kits 176 2110 176 2210 176 2310 176 2360 176 2410 176 2510 20 Description ProteOn GLC Sensor Chip for general amine coupling compact polymer layer with binding capacity of approximately one protein monolayer ProteOn GLM Sensor Chip for general amine coupling polymer matrix layer with intermediate binding capacity ProteOn GLH Sensor Chip for general amine coupling polymer matrix layer with highest binding capacity ProteOn NLC Sensor Chip for binding of biotinylated molecules contains NeutrAvidin immobilized to GLC layer ProteOn HTG Sensor Chip for capturing histidine tagged proteins polymer matrix layer contains tris NTA complexes with compact binding capacity ProteOn HTE Sensor Chip for capturing histidine tagged proteins polymer matrix layer contains tris NTA complexes with higher binding capacity ProteOn LCP Sensor Chip for capturing lipid assemblies such as liposomes for use with the ProteOn LCP capturing reagent kit ProteOn Liposome Capturing Kit includes 1 ProteOn LCP sensor chip 1 ProteOn LCP capturing reagent
20. ic acid Nucleic acid 5 10 mM NaOH deionized water HTX Sensor Chips The captured ligand can be stripped off and replaced with fresh ligand by a highly efficient regeneration step using 300 mM EDTA pH 8 5 Once the ligand is removed the chip can be reactivated to capture new ligands Table 9 HTX chip regeneration conditions Injection Reagent Orientation Volume ul Flow Rate pl min 1 300 mM EDTA pH 8 5 Vertical 400 30 EDTA may not completely remove nonspecifically adsorbed proteins because they are adsorbed to the surface not only via the nickel II ions but also by electrostatic interactions for example In such cases other ProteOn regeneration solutions such as 50 mM NaOH and 100 mM HCl may be needed along with EDTA to regenerate the surface LCP Chip and Liposome Capturing Kit Regeneration is accomplished by DNA dehybridization using the following conditions Injection 2 is optional because it is used to remove the remaining lipid assemblies if the regeneration is incomplete with injection 1 For the first injection use a freshly prepared 8 M solution of urea in deionized water Table 10 LCP chip regeneration conditions Injection Reagent Orientation Volume pl Flow Rate pl min 1 8 M urea Vertical 150 30 2 20 mM CHAPS Vertical 150 30 If urea is not available inject deionized water GLC Lipid Kit When using the GLC lipid kit injecting lipid modification solution 20 mM CHAPS will regenerate t
21. is taken out and reinserted in the instrument glycerol initialization must be used Setting Up a Protocol 1 Choose New or Open from the menu bar to open the database browser 2 Choose a Template Protocol or Experiment Edit the name as needed for your new experiment 3 In the Protocol screen edit the configuration samples and protocol steps as needed 4 In the Instrument Control screen set the chip temperature and sample temperature Running Experiments with Sensor Chips An interaction analysis experiment comprises five major steps 1 Conditioning 2 Ligand immobilization 3 Stabilization 4 Analyte injection 5 Regeneration Conditioning Chips Conditioning prepares the chip surface for use Although optional it is highly recommended because it can increase baseline stability The conditioning protocols for all sensor chips are listed in Table 1 Table 1 Conditioning parameters GLC GLM and GLH Chips Injection Reagent Orientation Volume yl Flow Rate pl min 1 0 5 SDS Horizontal 30 30 2 50 mM NaOH Horizontal 30 30 3 100 mM HCI Horizontal 30 30 4 0 5 SDS Vertical 30 30 5 50 mM NaOH Vertical 30 30 6 100 mM HCI Vertical 30 30 NLC Chip Injection Reagent Orientation Volume pl Flow Rate pl min il 50 mM NaOH Horizontal 30 30 2 1 M NaCl Horizontal 30 30 3 50 mM NaOH Vertical 30 30 4 1 M NaCl Vertical 30 30 HTG and HTE Chips Injection Reagent Orientation Volume yul
22. kit and ProteOn lipid modification conditioning solution ProteOn GLC Lipid Kit includes 1 ProteOn GLC sensor chip and 1 ProteOn lipid modification kit ProteOn HTG Capturing Kit includes 1 ProteOn HTG sensor chip and 1 ProteOn HTG and HTE reagent ki ProteOn HTE Capturing Kit includes 1 ProteOn HTE sensor chip and 1 ProteOn HTG and HTE reagent ki ProteOn Immobilization Buffer Kit includes 1 each sodium acetate buffer pH 4 0 4 5 5 0 5 5 ProteOn Regeneration and Conditioning Kit includes 1 each glycine buffer pH 1 5 2 0 2 5 3 0 and NaOH SDS HCI phosphoric acid NaCl 50 ml solution each ProteOn LCP Capturing Reagent Kit for capturing lipid assemblies such as liposomes for use with the ProteOn LCP sensor chip ProteOn Lipid Modification Kit includes ProteOn lipid modification conditioning solution and ProteOn lipid modification solution ProteOn Amine Coupling Kit includes EDAC EDC sulfo NHS and ethanolamine HCl ProteOn HTG and HTE Reagent Kit includes reagents for activation and regeneration of HTG and HTE sensor chips Related Literature Bio Rad 2009 How to perform excluded volume correction on the ProteOn XPR36 protein interaction system Bio Rad Bulletin 5822 Bio Rad 2011 Analyte interaction and kinetics Bio Rad Bulletin 6044 Bio Rad 2012 Reduction of nonspecific binding on ProteOn HTG and HTE sensor chips Bio Rad Bulletin 6302 Moriarty L 2010 Guide to Ligand Immobilization Bio
23. l contains the following information Expiration date Chip type Bar code Catalog number Storing Sensor Chips Store chips at 4 C To avoid condensation on the chip surface which can lead to inaccurate results keep sensor chips in the sealed nitrogen filled pouch during storage Sensor chips should also remain in the pouch until reaching room temperature before use Temperature equilibration takes from 30 to 60 min Opening a Sensor Chip 1 After temperature equilibration cut the top seal of the aluminum pouch 2 Hold the black end of the cartridge up inside the pouch to ensure the sensor chip slide remains inside the cartridge 3 Press the sensor chip slide firmly into place within the cartridge 4 Remove the sensor chip cartridge from the aluminum pouch Sensor chip slide Cartridge Initializing a Sensor Chip New sensor chips must be initialized in the instrument the first time they are used in an experiment Follow these steps to initialize the sensor chip using either air or glycerol 1 Insert the temperature equilibrated sensor chip into the instrument chip loader The chip ID chip chemistry and chip expiration date populate the Chip Details area of the Sensor Chip box in the ProteOn Manager software 2 Choose one of the initialization options using either air or glycerol 3 The additional Use Last initialization option is available for reuse of the sensor chip If the used sensor chip
24. ld be determined The desired immobilization level is calculated using the following equation M Png an 4R L R nax IS the desired maximum response when the ligand interacts with an analyte M is the molecular weight of the analyte M is the molecular weight of the ligand and n is the stoichiometric coefficient of the interaction the analyte ligand ratio For kinetic analysis aiming for an analyte response with Rmax lt 200 RU is recommended The approximate capacity of the amine coupling GLX chips the biotin capturing NLC chip and the histidine tag capturing HTX chips is as follows GLC 8kKRU GLM 12kRU GLH 20 kRU NLC 2kRU HTG 5kRU HTE 12 kRU Optimizing Immobilization Conditions In the ProteOn system the experimental conditions can be conveniently optimized by injecting reagents across multiple channels with each channel having different conditions for example varied concentrations of ligand and analyte GLX Sensor Chips Activation When using the amine coupling or GLX chips mix EDAC 1 ethyl 3 8 dimethylaminopropyl carbodiimide hydrochloride and sulfo NHS N hydroxysulfosuccinimide to produce the activation solution The activation reagents are typically prepared as a mixture of 1x EDAC sulfo NHS and should be mixed immediately before the injection We recommend using a contact time of 60 sec for moderate activation and 300 sec for high activation levels For the GLH chip avoid using a high
25. ne concentration by diluting the lipid modification solution undecylamine in the lipid modification conditioning solution CHAPS Liposome Capture Before liposome capture condition the chip surface with the lipid modification conditioning solution 20 mM CHAPS and then inject the liposome solution Table 5 Liposome capture parameters Injection Reagent Orientation Volume pl Flow Rate pl min 1 20 mM CHAPS Vertical 150 30 2 1 mg ml liposomes Vertical 150 30 When injecting ligand concentrations between 0 5 and 25 ug ml are typically used with a flow rate of 30 ul min and a contact time of 1 to 14 min These conditions will produce signals of up to 5 000 RU on the HTG chip and 12 000 RU on the HTE chip Please note that when capturing ligands from crude media various other proteins may also be adsorbed to the chip surface mainly due to nonspecific interaction with nickel Il ions Fortunately these proteins will not interfere with the kinetics because ligand analyte interactions are specific However the presence of nonspecifically bound lysate proteins will make accurate determination of ligand binding levels difficult given that the observed signal is the sum of the ligand signal and the signal from other bound proteins Troubleshooting Ligand Immobilization Amine Coupling with GLX Chips The degree of success in ligand immobilization can be visualized by observing the sensorgram during the procedure see Figu
26. ntibody NeutrAvidin NLC and LCP chips or a tris NTA complex HTG and HTE chips 3 Set the association time of the interaction to be long enough to observe curvature in the association phase Similarly allow the dissociation time of the interaction to be long enough to observe a signal drop in the dissociation phase The concentration range of analyte should span 10x above and 10x below the expected K A buffer blank can also be injected as a real time double row reference Note In protein small molecule interaction analysis the small molecule analyte is sometimes prepared with a high refractive index cosolvent such as DMSO In such a case excluded volume correction is typically used to account for the excluded volume effect while maintaining the accuracy of referencing Refer to bulletin 5822 for details on how to apply this correction Troubleshooting Analyte Injection Nonspecific Binding Nonspecific binding NSB is defined as the direct binding of an analyte or sample components other than the target ligand to the sensor chip surface NSB is characterized by significant binding responses that occur on reference spots and do not return to baseline at the end of the injection Figure 3 These events can potentially skew experimental results In theory if the NSB responses on the interaction surface and on the reference are similar subtracting the reference from the interaction response will correct the data and lead to accur
27. re 1 Typical Ligand Buffer Conditions The typical ligand buffer conditions are listed below 1 The pH should be one unit below the pl value 2 The ligand buffer ionic strength should be low and the ligand concentration should be above 0 1 ug ml 3 If a disulfide bond reduction reagent is needed in the ligand buffer TCEP is preferred over DTT because TCEP is compatible with the amine coupling protocol A B 25 0004 sede 5 20 000 Deactivation Deactivation T 15 0004 19 400 4 2 10 0004 Activation 13 800 a eee fe Activation Immobilization 4 8 200 4 8 5 000 Immobilization cc o 2 600 _ 5 000 r7 l l j S000 l l T T T 200 0 500 1000 1 500 200 180 560 940 1 320 1 700 Time sec Time sec Fig 1 Sensorgram examples of failed A and successful B ligand immobilization by amine coupling Activation Quality Verify the activation quality by 1 Using fresh activation reagents 2 Immobilizing another protein that was previously used under the same conditions with success Ligand Activity If a sufficiently high Rmax iS predicted based on the R value but the binding response is much lower than the expected response this indicates low ligand activity on the surface Because the amine coupling method randomly links any free amine group on the ligand molecule to the chip surface the ligand activity is usually not very high 1 If low ligand activity is caused by nonoptimized ligan
28. s This patented One shot Kinetics approach allows up to 36 individual interactions to be performed simultaneously in a single analyte injection providing high efficiency in experiment optimization and high throughput in data production Ligands and analytes are typically injected in the vertical and horizontal directions respectively For kinetic analysis analyte injections are usually performed at a high flow rate for example 100 ul min but a lower flow rate may be used to reduce sample consumption The injection conditions including association and dissociation time flow rate and analyte concentrations should be optimized to obtain high quality interaction analysis Refer to bulletin 6044 for further details on achieving optimal ligand analyte interaction analysis Three useful tips for obtaining reliable interaction analysis results are listed below 1 Prepare the analyte samples as a concentration series typically a two or threefold dilution series centered around the expected Kp 2 If needed set up a double reference Replace one of the six analyte channels with running buffer for use as a real time double reference row reference Alternatively set up an injection of running buffer into all six analyte channels prior to the injection of analyte samples injection reference Double referencing is needed for baseline drift correction and is used mostly when the ligand is reversibly captured by a capture reagent such as an a
29. t reduce NSB directly but it shields the charges on the chip surface as much as the ligand NSB will be corrected for by subtracting the reference Dilute the When using a complex analyte analyte sample like serum or crude lysate dilute the sample with the running buffer a five to tenfold dilution e e is usually recommended Higher dilution rates should be used if the sample is very concentrated Use Use simple prepurification methods prepurification to remove the majority of the e contaminants in the analyte sample Remove If a ligand sample contains albumin from albumin inject a pulse of 1 M the ligand salt solution to remove the a e sample albumin bound to the chip surface before the analyte injection Note Refer to bulletin 6302 for specific troubleshooting tips for HTX chips Bulk Effect The bulk effect refers to a spurious SPR response that is caused by changes in the refractive index of the solution near the sensing surface rather than the binding of biomolecules to the surface Such refractive index changes typically occur during the sequential injection of two solutions with different compositions such as different salt detergent or biomolecule concentrations A small bulk effect can be completely removed by applying proper referencing but a large bulk effect may cause inaccuracy in data processing and analysis To minimize the bulk effect the refractive index of the running buffer should match that of the sample
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