MSIA Protein A, G and A/G Tips Technical Manual
Contents
1. Cat No Description 650 01 BS Versette Base Unit Stage Head Housing and Pipetting Head Required for Use 650 02 NTC 96 and 384 Channel Housing Assembly For Use with 96 and 384 Channel Pipetting Heads 650 03 SPS 6 Position Stage Guarding Included 650 06 96300 96 Channel Air Displacement Pipetting Head Volume 5 300 ul 650 04 PUMP Pump Module Optional Accessory Used for Tip Washing Reagent Replenishing 650 05 96TTW 96 Channel Tip Wash Station Tall Optional Accessory 650 08 96300SD Serial Dilute Magazine 96 300 ul 8 12 Multichannel Pipettes and Pipette Stand Description Finnpipette Novus i Electronic 8 Channel Pipette 20 300 pl for immuno precipitation Quantity 1 pipette Finnpipette Novus i Electronic 12 Channel Pipette 20 300 pl for immuno precipitation 1 pipette Finnpipette Novus i Adjustable Pipette Stand for immuno precipitation 1 pipette stand Finnpipette Novus i Electronic 8 Channel Pipette 20 300 pl and Pipette Stand for immuno precipitation 1 pipette and stand Finnpipette Novus i Electronic 12 Channel Pipette 20 300 ul and Pipette Stand for immuno precipitation 1 pipette and stand Liquid Chromatography Description UltiMate 3000 RSLCnano Systems Mass Spectrometry and Software Mass Spectrometer and Software Description TSQ Vantage Triple Stage Quadrupole Mass Spectrometer Pinpoint Software Q Exa2. Thermo Scientific MSIA Protein A G and A G Tips Thermo Scientific Protein A G and A G MSIA Tips Products Information MSIA D A R T S Pipette Tips Compatible with the Versette Automated Liquid Platform Finnpipette Novus i Multichannel Electronic Pipettes for immuno precipitation also with select Eppendorf Biohit and Hamilton Multichannel Pipettes Cat No Description Packaging 991PRT11 300 ul MSIA D A R T S Protein A Pack of 96 tips 991PRT12 300 ul MSIA D A R T S Protein A Pack of 24 tips 991PRT13 300 ul MSIA D A R T S Protein G Pack of 96 tips 991PRT14 300 ul MSIA D A R T S Protein G Pack of 24 tips 991PRT15 300 ul MSIA D A R T S Protein A G Pack of 96 tips 991PRT16 300 ul MSIA D A R T S Protein A G Pack of 24 tips MSIA Pipette Tips Compatible to Beckman Multimek Type II Liquid Handling System Cat No Description Packaging 991PRTO1 200 ul MSIA Tips Protein A Pack of 96 tips 991PRTO2 200 ul MSIA Tips Protein G Pack of 96 tips 991PRTO3 200 ul MSIA Tips Protein A G Pack of 96 tips Storage Upon receipt store at 4 C Product shipped with an ice pack Disclaimer These products are supplied for life science research use only They are not intended for medicinal diagnostic or therapeutic use Table of Content Important product information 3 Protein A MSIA Tips protocol 4 7 Pr
3. A measured aliquot of the analytical sample is then dispensed into either an individual well of a micro titer plate or a micro centrifuge tube Notes e Sample volume and concentration may be modified according to the researcher preference The sample dilution and volume are dictated by the biological matrix selected as well as the concentration of the analyte being targeted If the analytical sample amount required exceeds the volume of the micro titer plate or micro centrifuge tubes described in this protocol then appropriately sized receptacles may be substituted e Cycle volume and number of cycle iterations may be modified according to the researcher preference If the Novus i electronic pipette is used we recommend starting with speed level 5 The speed of the Novus i pipette cycling can be further optimized by the researcher Studies should be performed by researcher to ensure optimal extraction purification of the targeted analyte by the MSIA Tips e Ifthe samples are frozen or refrigerated they must be thawed and or warmed to at least room temperature before preparation A 37 C water bath may be employed to expedite this process and ensure consistent sample temperature The raw sample should also be centrifuged prior to aliquot distribution in order to ensure the removal of any particulates or debris that may be present e Jf serial dilutions are required to prepare the analytical sample use the Wash Buffer for each step and thoroughly m
4. also readily automated with liquid handling robots such as theThermo Scientific Versette Liquid Handling Platform which is especially useful for large scale screening of multiple samples Protein A MSIA Tips contain a covalently immobilized recombinant Protein A 44 600 Da apparent molecular weight by SDS PAGE 45KDa that is expressed in E coli and functions essentially the same as native Protein A Protein A contains four Fc binding domains that potentially interact with immunoglobulins The interaction of Protein A with immunoglobulins is pH dependent with the optimal condition being pH 8 2 although binding is good at neutral or physiological pH pH 7 0 7 6 Furthermore this interaction is not equivalent for all species nor is it within species i e the interaction is stronger with some immunoglobulin subclasses than others Protein G MSIA Tips contain a covalently immobilized recombinant Protein G 21 600 Da apparent molecular weight by SDS PAGE 32KDa that enables the probing and detection of mouse and human antibodies especially IgG isotypes In addition the albumin and cell surface binding domains of Protein G have been eliminated in the recombinant Protein G to reduce nonspecific binding and therefore can be used to separate immunoglobulins from crude samples Recombinant Protein G contains two Fc binding domains that can interact with immunoglobulins The optimal pH for Protein G to bind immunoglobulins is pH 5 However effective bi
5. can be prepared in bulk and stored at 4 C until use Buffer must be warmed to room temperature prior to use The prepared antibody solution used in the loading phase should be thoroughly mixed in the Buffer by repeat inversion or using a rotating platform Aggressive agitation of antibody solution will result in the denaturing of the antibody Prepare the antibody solution for use in loading The antibody will be prepared in the Dilution Buffer at a final concentration range of 0 01 0 1 mg mL Add 100 uL of this antibody solution into each well of the micro titer plate or micro centrifuge tube A single well or tube of antibody solution is necessary for each Protein G MSIA Tip to be loaded For each Protein G MSIA Tip to be loaded a well or micro centrifuge tube of each Dilution Buffer and Wash Buffer 200 uL are also required The general workflow for antibody loading is presented in Table 1 These are general conditions and should be optimized by the researchers The pipetting cycle iterations are performed by repetitively aspirating and dispensing standard pipette mixing action a cycle 1 aspiration and 1 dispense using the listed cycle volumes provided The numbers of iterations provided are a general guideline and may require further optimization by the researcher Protein G MSIA Tips Table 1 Antibody Load Workflow Steps Description Cycle Volume No Cycle Approx Allotted L Iterations time per step 1 Wash Buffer 150 10 1 minu
6. costs of investigating and responding to such request at Seller s then prevailing time and materials rates If Seller provides repair services or replacement parts that are not covered by this warranty Buyer shall pay Seller therefore at Seller s then prevailing time and materials rates Any installation maintenance repair service relocations or alteration to or of or other tampering with the products preformed by any person or entity other than seller without sellers s prior written approval or any use of replacement parts not supplied by seller shall immediately void and cancel all warranties with respect to the affected products The obligations created by this warranty statement to repair or replace a defective product shall be the sole remedy of buyer in the event of defective product except as expressly provided in this warranty statement seller disclaims all other warranties whether express or implied oral or written with respect to the products including without limitations all implied warranties of merchantability or fitness for any particular purpose Seller does not warrant that the products are error free or will accomplish any particular result 16 thermoscientific com msia 2012 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Pl
7. e Protein A G has a broader binding range than either Protein A or Protein G individually Protein A G binds to all human IgG subclasses binds somewhat to IgA IgE IgM and to a lesser extent IgD Unlike Protein G Protein A G does not bind serum albumin because the gene sequence coding for the albumin binding site has been eliminated Protein A G is effective for use with mouse monoclonal antibodies and binds to all mouse IgG subclasses but not IgA IgM or serum albumin e For more information see Tech Tip 34 Binding Characteristics for Immunoglobulins and Protein L A G and A G at www thermoscientific com pierce Protein A MSIA Tips Procedure for Protein A Antibody Loading Note The following information provided in this procedure describes the use of the Thermo Scientific Protein A MSIA Tips in single manual pipette application format The steps described are readily translatable to multi channel and robotic platform applications however they are not described in this section This procedure is for general guidance only and may require further optimization by the researcher Additional Materials Required Thermo Scientific Finnpipette Novus i Electronic Multichannel Pipette and adjustable pipette stand or Thermo Scientific Versette Liquid Handling Platform or the appropriate Beckman liquid handling platform 96 well microplate U bottom polypropylene Thermo Scientific Product No 267334 or 0 6 mL Fisherbrand Snap Cap Flat Top Grad
8. are the antibody solution for use in loading The antibody should be prepared in the Dilution Buffer at a final concentration range of 0 01 0 1 mg mL Add 100 uL of this antibody solution into each well of the micro titer plate or micro centrifuge tube A single well or tube of antibody solution is necessary for each Protein A G MSIA Tips to be loaded For each Protein A G MSIATips to be loaded a well or micro centrifuge tube of Wash Buffer 200 uL is also required The general workflow for antibody loading is presented in Table 1 These are general conditions and should be optimized by the researchers The pipetting cycle iterations are performed by repetitively aspirating and dispensing standard pipette mixing action a cycle 1 aspiration and 1 dispense using the listed cycle volumes provided The numbers of iterations provided are a general guideline and may require further optimization by the researcher Protein A G MSIA Tips Table 1 Antibody Load Workflow 12 Steps Description Cycle Volume No Cycle Approx Allotted uL Iterations time per step 1 Wash Buffer 150 10 1 minute 2 Antibody Solution 75 250 gt 15 minutes 3 Wash Buffer 150 10 1 minute The workflow is performed by loading the Protein A G MSIA Tips onto a pipette and immersing the open end of the tip into the volume of liquid for each of the Steps provided Perform each cycle using the normal pipetting action as specified in the pipette user manual Zf the Novus i e
9. arts may be new or refurbished at the election of Seller All replaced parts shall become the property of Seller Shipment to Buyer repaired or replacement Products shall be made in accordance with the Delivery provisions of the Seller s Term and Conditions of Sale Consumables are expressly excluded this warranty Notwithstanding the foregoing Products supplied by Seller that are obtained by Seller from an original manufacturer or third party supplier are not warranted by Seller but Seller agrees to assign to Buyer any warranty rights in such Product that Seller may have from the original manufacturer or third party supplier to the extent such assignment is allowed by such original manufacturer or third party supplier In no event shall Seller have any obligation to make repairs replacements or corrections required in whole or in part as the result as i normal wear and tear ii accident disaster or event of force majeure iii misuse fault or negligence of or by Buyer iv use of the Products in a manner for which they were not designed v causes external to the Products such as but not limited to power failure or electrical power surges vi improper storage and handling of the Products or vii use of the Products in combination with equipment or software not supplied by Seller If Seller determines that Products for which Buyer has requested warranty services are not covered by the warranty hereunder Buyer shall pay or reimburse Seller for all
10. ctive Hybrid Quadrupole Orbitrap Mass Spectrometer Warranty Sellers warrants that the Products will operate or perform substantially in conformance with Seller s published specifications and be free from defects in material and workmanship when subjected to normal proper and intended usage by properly trained personnel for the period of time set forth in the product documentation published specifications or page insert If a period of time is not specified in Seller s product documentation published specifications or package inserts the warranty period shall be one 1 year from the date of shipment to Buyer for equipment and ninety 90 days for all other products the Warranty Period Seller agrees the Warranty Period to repair or replace at Seller s option defective Products so as to cause the same to operate in substantial conformance with said published specifications provided that Buyer shall a promptly notify Seller in writing upon the discovery of any defect which notice shall include the product model and serial number if applicable and details of the warranty claim and b after Seller s review Seller will provide Buyer with service data and or a Return Material Authorization RMA which may include biohazard decontamination procedures and other product specific handling instructions then if applicable Buyer may return the effective Products to Seller with all costs prepaid by Buyer Replacement p
11. ease consult your local sales representative for details North America 1 800 995 2787 infosandiego thermofisher com Outside North America 1 858 453 7551 info sandiego thermofisher com The SCIENTIFIC Part of Thermo Fisher Scientific 407224 RevA
12. general protocols for cross linking antibodies to protein A C Sample Analyte Loading 1 The analytical sample of choice is prepared by dispensing a measured aliquot of analyte containing biological matrix into a fixed volume of Dilution Buffer The diluted sample is then thoroughly mixed via repeat inversion vortexing or rotary platform A measured aliquot of the analytical sample is then dispensed into either an individual well of a micro titer plate or a micro centrifuge tube Notes e Sample volume and concentration can be modified according to the researcher s preference The sample dilution and volume are dictated by the biological matrix selected as well as the concentration of the analyte being targeted If the analytical sample amount required exceeds the volume of the micro titer plate or micro centrifuge tubes described in this protocol then appropriately sized receptacles may be substituted e Cycle volume and number of cycle iterations may be modified according to the researcher preference If the Novus i electronic pipette is used we recommend starting with speed level 5 The speed of the Novus i pipette cycling can be further optimized by the researcher Studies should be performed by researcher to ensure optimal extraction purification of the targeted analyte by the MSIA Tips e Ifthe samples are frozen or refrigerated they must be thawed and or warmed to at least room temperature before preparation A 37 C water bath may be em
13. ix prior to the next dilution in the same manner as described above Protein G MSIA Tips 2 For each Protein G MSIA Tip used a well or micro centrifuge tube of Wash Buffer 200 uL and two corresponding wells tubes of water 200 uL per are also required 3 The mechanical application of the Protein G MSIA Tip in sample analyte loading is exactly the same as previously described for the antibody loading The general workflow of the sample loading is presented in Table 2 Table 2 Sample Analyte Load Workflow Steps Description Cycle Volume No Cycle Approx Allotted uL Iterations time per step 1 Wash Buffer 150 10 1 minute 2 Analytical Sample 100 150 100 gt 5 minutes 3 Wash Buffer 150 10 1 minute 4 Water 150 10 1 minute 5 Water 150 10 1 minute Note The general workflow provided is to serve as a starting point The rinse steps number of cycle iterations etc can all be tailored to the researcher application 4 Once the Protein G MSIA Tip is loaded with target analyte it is ready for subsequent elution and any post elution treatment if necessary Subsequent steps are specific for the application and the analytical detection system selected by researcher 10 11 Protein A G MSIA Tips Procedure for Protein A G Antibody Loading Note The following information provided in this procedure describes the use of the Protein A G MSIA Tips in single manual pipette application format The steps described are readily translatable
14. lectronic pipette is used we recommend starting with speed level 5 The speed of the Novus i pipette cycling can be further optimized by the researcher Note To maintain proper sample and wash flow through the Protein A G MSIA Tips ensure that the open end remains completely immersed in the volume of liquid during each step of the workflow Failure to do so will result in aspiration of air and inconsistency in liquid movement within the tip Also do not press the Tip into the bottom of the liquid holding vessel as this will restrict flow Once the Protein A G MSIA Tip is loaded with antibody it may be immediately used for analyte purification or stored dry at 4 C for later use Furthermore the captured antibody may also be cross linked to the protein A G following general protocols for cross linking antibodies to protein A G Sample Analyte Loading The analytical sample of choice is prepared by dispensing a measured aliquot of analyte containing biological matrix into a fixed volume of Dilution Buffer The diluted sample is then thoroughly mixed via repeat inversion vortexing or rotary platform A measured aliquot of the analytical sample is then dispensed into either an individual well of a micro titer plate or a micro centrifuge tube Notes e Sample volume and concentration may be modified according to the researcher preference The sample dilution and volume are dictated by the biological matrix selected as well as the concentration
15. nding can be achieved at pH 7 0 7 2 Protein A G MSIA Tips contain a covalently immobilized recombinant Protein A G 50 500 Da apparent molecular weight by SDS PAGE 40 45K that combines the IgG binding domains of both Protein A and Protein G Protein A G contains 4 Fc binding domains from Protein A and two from Protein G making it a more general and convenient tool for investigating and purifying immunoglobulins Also Protein A G binding to immunoglobulins is not as pH dependent as Protein A Important Product Information e Store the Protein A G and A G MSIA Tips at 4 C do not freeze e This product is intended for single use only e Thermo Scientific Protein A G and A G MSIA Tips are not intended for the transference or measurement of liquids This product is intended for micro scale analyte purification prior to mass spectrometric detection e Protein A binds strongly to human subclasses IgG IgG and IgG4 but does not bind to IgG In many instances Protein A does not bind to monoclonal antibodies especially those produced in rat or from mouse that are of the IgG subclass e Protein G has greater affinity than Protein A for most mammalian IgGs and may be used for the purification of mammalian IgGs that do not bind well to Protein A Protein G binds with significantly greater capacity than Protein A to several IgG subclasses such as human IgG3 mouse IgG1 and rat IgG2a However Protein G does not bind to human IgM IgD and IgA
16. nse using the listed cycle volumes provided The numbers of iterations provided are a general guideline and may require further optimization by the researcher Protein A MSIA Tips Table 1 Antibody Load Workflow Step Description Cycle Volume No Cycle Approx Allotted uL Iterations time per step 1 Wash Buffer 150 10 1 minute 2 Antibody Solution 75 gt 250 gt 15 minutes 3 Wash Buffer 150 10 1 minute The workflow is performed by loading the Protein A MSIA Tip onto a pipette and immersing the open end of the tip into the volume of liquid for each of the steps provided Perform each cycle using the normal pipetting action as specified in the pipette user manual Jf the Novus i electronic pipette is used we recommend starting with speed level 5 The speed of the Novus i pipette cycling can be further optimized by the researcher Note To maintain proper sample and wash flow through the Protein A MSIA Tip ensure that the open end remains completely immersed in the volume of liquid during each step of the workflow Failure to do so will result in aspiration of air and inconsistency in liquid movement within the tip Also do not press the Tip into the bottom of the liquid holding vessel as this will restrict flow 4 Once the Protein A MSIA Tip is loaded with antibody it may be immediately used for analyte purification or stored dry at 4 C for later use Furthermore the captured antibody may also be cross linked to the protein A following
17. of the analyte being targeted If the analytical sample amount required exceeds the volume of the micro titer plate or micro centrifuge tubes described in this protocol then appropriately sized receptacles may be substituted e Cycle volume and number of cycle iterations may be modified according to the researcher preference If the Novus i electronic pipette is used we recommend starting with speed level 5 The speed of the Novus i pipette cycling can be further optimized by the researcher Studies should be performed by researcher to ensure optimal extraction purification of the targeted analyte by the MSIA Tips e If the samples are frozen or refrigerated they must be thawed and or warmed to at least room temperature before preparation A 37 C water bath may be employed to expedite this process and ensure consistent sample temperature The raw sample should also be centrifuged prior to aliquot distribution in order to ensure the removal of any particulates or debris that may be present e If serial dilutions are required to prepare the analytical sample use the Dilution Buffer for each step and thoroughly mix prior to the next dilution in the same manner as described above 2 Protein A G MSIA Tips For each Protein A G MSIA Tips used two wells or micro centrifuge tubes of Wash Buffer 200 uL and two corresponding wells tubes of water 200 uL per are also required The mechanical application of the Protein A G MSIA Tips in sample analy
18. or the application and the analytical detection system selected by researcher Protein G MSIA Tips Procedure for Protein G Antibody Loading Note The following information provided in this procedure describes the use of the Protein G MSIA Tips in single manual pipette application format The steps described are readily translatable to multi channel and robotic platform applications however they are not described in this section This procedure is for general guidance only and may require further optimization by the researcher A Additional Materials Required Thermo Scientific Finnpipette Novus i Electronic Multichannel Pipette and adjustable pipette stand or Thermo Scientific Versette Liquid Handling Platform or the appropriate Beckman liquid handling platform 96 well microplate U bottom polypropylene Thermo Scientific Product No 267334 or 0 6 mL Fisherbrand Snap Cap Flat Top Graduated Microcentrifuge Tubes Fisher Scientific Product No 02 681 257 Purified Water Dilution Buffer 10mM MES pH 5 MES Fisher Scientific Product No BP300 100 containing 0 1 final concentration Tween 20 Detergent Thermo Scientific Product No TA 125 TW Wash Buffer Phosphate buffered saline PBS Thermo Scientific Product No 28372 or Tris buffered saline TBS Thermo Scientific Product No 28379 All wash buffers prepared also include 0 1 final concentration Tween 20 Detergent Antibody Loading Notes Diluent Wash Buffer
19. otein G MSIA Tips protocol 8 10 Protein A G MSIA Tips protocol 11 13 Additional information 14 MSIA related Thermo Scientific products 15 Warranty 16 Introduction The Mass Spectrometric Immunoassay MSIA Tips provide a fast convenient and highly reproducible method for both manual and automated enrichment of target analytes for subsequent mass spectrometric detection Thermo Scientific Protein A MSIA Tips provide the researcher with the flexibility to tailor the devices for specific target analyte applications by using their own antibodies Such immunoaffinity enrichment is typically used for isolating analytes from but not limited to serum plasma urine or cell culture supernatants For immuno affinity capture and enrichment of desired target analytes the protein A G and A G MSIA Tips previously loaded with antibody are used to interrogate a prepared biological sample by employing a cyclical pipetting motion This allows for simultaneous purification and enrichment of the targeted analyte After sample incubation the tips are rinsed in the same fashion and the bound antigens are then dissociated from the tips using an elution buffer The elution buffer selection and the methodologies for mass spectrometric detection are both application and target specific Manual purification 8 12 samples can be performed using the Thermo Scientific Finnpipette Novus i electronic multichannel pipette and adjustable pipette stand The method is
20. ployed to expedite this process and ensure consistent sample temperature The raw sample should also be centrifuged prior to aliquot distribution in order to ensure the removal of any particulates or debris that may be present e Tf serial dilutions are required to prepare the analytical sample use the Dilution Buffer for each step and thoroughly mix prior to the next dilution in the same manner as described above 2 For each Protein A MSIA Tip used a well or micro centrifuge tube of Wash Buffer 200 uL and two corresponding wells tubes of water 200 uL per are also required 3 The mechanical application of the Protein A MSJA Tip in sample analyte loading is exactly the same as previously described for the antibody loading The general workflow of the sample loading is presented in Table 2 Protein A MSIA Tips Table 2 Sample Analyte Load Workflow Step Description Cycle Volume No Cycle Approx Allotted uL Iterations time per step 1 Wash Buffer 150 10 1 minute 2 Analytical Sample 100 150 gt 100 gt 5 minutes 3 Wash Buffer 150 10 1 minute 4 Water 150 10 1 minute 5 Water 150 10 1 minute Note The general workflow provided is to serve as a starting point The rinse steps number of cycle iterations etc can all be tailored to the researcher s application 4 Once the Protein A MSIA Tip is loaded with target analyte it is ready for subsequent elution and any post elution treatment if necessary Subsequent steps are specific f
21. se go to www thermoscientific com or your local distributor Thermo Fisher Scientific Inc 09 2012 All rights reserved Eppendorf is a registered trademark of Eppendorf AG Biohit is a trademark of Biohit Oy Hamilton is a trademark of Hamilton Robotics Beckman is a registered trademark of Beckman Coulter All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details Printed in the USA 14 MSIA related Thermo Scientific Products 15 MSIA D A R T S Pipette Tips Compatible with the Versette Automated Liquid Platform Finnpipette Novus i Multichannel Electronic Pipettes for immuno precipitation also with select Eppendorf Biohit and Hamilton Multichannel Pipettes Cat No Description 991PRT11 300 ul MSIA D A R T S Protein A 991PRT12 300 ul MSIA D A R T S Protein A 991PRT13 300 ul MSIA D A R T S Protein G 300 ul MSIA D A R T S Protein G Pack of 24 tips 300 ul MSIA D A R T S Protein A G Pack of 96 tips 300 ul MSIA D A R T S Protein A G Pack of 24 tips 300 ul MSIA D A R T S Custom Pack of 96 tips 300 ul MSIA D A R T S Reloadable Rack 1reloadable rack tips are not Automated Liquid Handling Platform and Pipetting Head
22. te 2 Antibody Solution 75 250 gt 15 minutes 3 Wash Buffer 150 10 1 minute The workflow is performed by loading the Protein G MSIA Tip onto a pipette and immersing the open end of the tip into the volume of liquid for each of the Steps provided Perform each cycle using the normal pipetting action as specified in the pipette user manual Zf the Novus i electronic pipette is used we recommend starting with speed level 5 The speed of the Novus i pipette cycling can be further optimized by the researcher Note To maintain proper sample and wash flow through the Protein G MSIA Tip ensure that the open end remains completely immersed in the volume of liquid during each step of the workflow Failure to do so will result in aspiration of air and inconsistency in liquid movement within the tip Also do not press the Tip into the bottom of the liquid holding vessel as this will restrict flow 3 Once the Protein G MSIA Tip is loaded with antibody it may be immediately used for analyte purification or stored dry at 4 C for later use Furthermore the captured antibody may also be cross linked to the protein G following general protocols for cross linking antibodies to protein G C Sample Analyte Loading 1 The analytical sample of choice is prepared by dispensing a measured aliquot of analyte containing biological matrix into a fixed volume of Wash Buffer The diluted sample is then thoroughly mixed via repeat inversion vortexing or rotary platform
23. te loading is exactly the same as previously described for the antibody loading The general workflow of the sample loading is presented in Table 2 Table 2 Sample Analyte Load Workflow 13 Steps Description Cycle Volume No Cycle Approx Allotted uL Iterations time per step 1 Wash Buffer 150 10 1 minute 2 Analytical Sample 100 150 gt 100 gt 5 minutes 3 Wash Buffer 150 10 1 minute 4 Water 150 10 1 minute 5 Water 150 10 1 minute Note The general workflow provided is to serve as a starting point The rinse steps number of cycle iterations etc can all be tailored to the researcher application Once the Protein A G MSIA Tips are loaded with target analyte it is ready for subsequent elution and any post elution treatment if necessary Subsequent steps are specific for the application and the analytical detection system selected by researcher Additional Information Visit the www thermoscientific com msia for additional information and application notes relating to this product Visit www thermoscientific com pierce for information specifically related to the Protein A G and A G e Tech Tip 34 Binding Characteristics of Immunoglobulins and Protein L A G and A G Product stable for one year from date of sale when handled and stored according to Manufacturer instructions see details under Warranty The most current versions of all MSIA product instructions are available at www thermoscientific com msia For a copy plea
24. to multi channel and robotic platform applications however they are not described in this section This procedure is for general guidance only and may require further optimization by the researcher Additional Materials Required Thermo Scientific Finnpipette Novus i Electronic Multichannel Pipette and adjustable pipette stand or Thermo Scientific Versette Liquid Handling Platform or the appropriate Beckman liquid handling platform 96 well microplate U bottom polypropylene Thermo Scientific Product No 267334 or 0 6 mL Fisherbrand Snap Cap Flat Top Graduated Microcentrifuge Tubes Fisher Scientific Product No 02 681 257 Purified Water Dilution Buffer 10mM MES pH 5 MES Fisher Scientific Product No BP300 100 containing 0 1 final concentration Tween 20 Detergent Thermo Scientific Product No TA 125 TW Wash Buffer Phosphate buffered saline PBS Thermo Scientific Product No 28372 or Tris buffered saline TBS Thermo Scientific Product No 28379 All wash buffers prepared also include 0 1 final concentration Tween 20 Detergent Antibody Loading Notes Diluent Wash Buffer can be prepared in bulk and stored at 4 C until use Buffer must be warmed to room temperature prior to use The prepared antibody solution used in the loading phase should be thoroughly mixed in the Buffer by repeat inversion or using a rotating platform Aggressive agitation of antibody solution will result in the denaturing of the antibody Prep
25. uated Microcentrifuge Tubes Fisher Scientific Product No 02 681 257 Purified Water Dilution Wash Buffer Phosphate buffered saline PBS Thermo Scientific Product No 28372 or Tris buffered saline TBS Thermo Scientific Product No 28379 All buffers prepared also include 0 1 final concentration Tween 20 Detergent Thermo Scientific Product No TA 125 TW Antibody Loading Notes Diluent Wash Buffer can be prepared in bulk and stored at 4 C until use Buffer must be warmed to room temperature prior to use The prepared antibody solution used in the loading phase should be thoroughly m ixed in the Buffer by repeat inversion or using a rotating platform Aggressive agitation of antibody solution will denature the antibody Prepare the antibody solution for use in loading The antibody should be prepared in the Dilution Buffer at a final concentration in the range of 0 01 0 1 mg mL Add 100 uL of this antibody solution into each well of the micro titer plate or micro centrifuge tube A single well or tube of antibody solution is necessary for each Protein A MSIA Tip to be loaded For each Thermo Scientific Protein A MSIA Tip to be loaded a well or micro centrifuge tube of Wash Buffer 200 uL is also required The general workflow for antibody loading is presented in Table 1 The pipetting cycle iterations are performed by repetitively aspirating and dispensing standard pipette mixing action a cycle 1 aspiration and 1 dispe
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