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1. I I gd 220 240 260 200 300 320 340 360 380 400 420 440 460 460 500 520 550 Stee An Wavelength nm Figure 9 NanoDrop absorption spectrum of unmodified horseradish peroxidase 220 550 nm 0 66 mg ml sodium phosphate buffer pH 6 0 1 mm path length F 4FB modified HRP Absorption Spectrum 4FB HRP 0 66 mg ml Sample 18 Baseline 0 000 A1 280 nm Abs 1 0040 1 mm Absorbance 42 403 Z nm Abs 2 0065 1 03 I I l l l l l l l l l l l I I a 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 4550 Wavelength nm Figure 10 NanoDrop absorption spectrum of 4FB modified horseradish peroxidase 220 550 nm 0 66 mg ml sodium phosphate buffer pH 6 0 1 mm path length 23 G Bovine IgG HRP Conjugate Absorption Spectrum All in One Purified ampie IL Mouse HRP IgG Sample 6 Baseline 0 000 0 280 lt nm 0 195 a O c m E o w 2 4 E E 40 nm 0 101 hy zd bo zda slo lb io sb ob slo xU ak so ah sk 91 NKA ENAR Wavelength nm Figure 11 NanoDrop absorption spectrum of All in One IgG HRP conjugate 220 550 nm lt 2 0 96 mg ml sodium phosphate buffer pH 6 0 1 mm path length H Concentration of Dilute Antibody Solutions The HRP Antibody All in One Conjugation protocol reguires that initial antibody protein concentration be at 4 5 mg ml and 25 ul Many antibody vendors package their products at significantly more dilute concentrations e g
2. 0 25 to 1 5 mg ml In these instances IgG samples will need to be concentrated to 4 5 mg ml and 25 ul before proceeding The All in One kit provides two 2 diafiltration filters M W C O 30 kD for this purpose Figure 12 Carefully follow these instructions to avoid antibody loss or aggregation on the filter s surface Note Dilute antibody solutions require at least 125 jug of starting antibody e g 500 ul 0 25 mg ml since diafiltration filters recover 80 of input antibody If antibody samples are not inlimiting supply we recommend antibody concentrations be confirmed using a Bradford protein assay before proceeding lt Y Concentrator body ww x m m Filtrate tube Figure 12 Diafiltration spin filter used for concentrating dilute antibody samples prior to the start of All in One conjugation protocol 24 NAN o Antibody Concentration Protocol Note the diafiltration spin filters illustrated is made to contain and process a maximum volume of 500 ul or less If a volume greater than 500 ul is to be concentrated multiple loadings will be required Open the lid of a diafiltration spin filter device Transfer 500 ul or less of dilute protein solution equivalent to 125 ug antibody to the center of the filter cup Close the lid and orient the spin filter in the centrifuge so that the volume markers face toward the center of the centrifuge rotor Use an appropriate ba
3. aiming outward and away from the center of the rotor Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided Remove the red cap load the antibody sample 25 uL at 4 mg mL to the top of the dry resin bed loosely recap and place the column back into the collection tube Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes Use an appropriate balance tube opposite the assembly Important Rotor speed must be set to 1500 x g RCF and not 1500 x rom RPM The volume recovered should always be approximately the same volume loaded onto the spin column e g 25 5 uL If the recovered volume is low the centrifuge may require recalibration If recovered volume is low re centrifuge at the appropriate speed in an attempt to recover the full volume i e 25 uL Transfer the buffer exchanged IgG solution 25 30 pL from the bottom of the collection tube to a new 1 5 mL tube and label appropriately C HyNic Modify IgG 2 h 1 Add 20 ul DMF to the vial of S HyNic reagent Pipette the solution up and down to resuspend the reagent pellet Note a small but visible pellet can be seen at the bottom of the vial Add 1 5 ul dissolved S HyNic reagent to the antibody solution 25 ul 4 mg mL Pipette the solution up and down to mix Incubate the reaction
4. sample appropriately e g HyNic IgG E Conjugate Formation 2 h 1 Briefly spin the dark brown vial containing 4FB modified HRP 5 seconds 1000 x g to collect the contents at the bottom of the tube Transfer 50 ul 4FB modified HRP to the tube containing HyNic modified antibody 25 35 uL pipette up and down to mix Incubate the reaction for 2 h at room temperature to form the conjugate 13 F Buffer Exchange Conjugate 3 minutes 1 After completion of the conjugation reaction prepare a spin column brown cap by twisting off the bottom closure and loosening the brown cap do not remove Place the spin column into a collection tube provided Remember equilibrate all kit components to ambient room temperature before use 2 Mark the top of the brown cap using an indelible pen to identify the sample Also place a vertical mark on the side of each spin column as shown below Label lid w conjugate ID T Place pen mark on side of spin column Collection tube 3 Place the assembly into the centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor Use an appropriate balance tube opposite the spin filter 4 Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided 5 Open the brown cap load 75 90 u
5. 870 S HyNic 1 yr Keep in sealed aluminum pouch provided 2 8 C 24 h after re suspending Room temperature S HyNic in DMF HRP Antibody 9 month Refrigerated 2 8 C in Conjugate final conjugate solution 1 yr Refrigerated 2 8 C 50 glycerol All other kit components Refrigerated 2 8 C Flash Drive Room temperature 27 References Dirksen A Hackeng T Dawson P 2007 Nucleophilic Catalysis of Oxime and Hydrazone Reactions by Aniline ACS Poster Dirksen A Hackeng T Dawson P 2006 Nucleophilic Catalysis of Oxime Ligations Angew Chem Int Ed 45 7581 7584 Dirksen A Dirksen S Hackeng T Dawson P 2006 Nucleophilic Catalysis of Hydrazone Formation and Transimination Implications for Dynamic Covalent Chemistry JIAICIS Communications S Lim H P Manusu A A Gooley K L Williams and D B Rylatt Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis Journal of Chromatography A Vol 827 Issue 2 11 December 1998 Pages 329 335 Francesca Chiodi Dr Ake Sid n Eva Osby 2005 Isoelectric focusing of monoclonal immunoglobulin G A and M followed by detection with the avidin biotin system Electrophoresis Vol 6 Issue 3 124 128 28
6. L conjugate solution section e step 3 to the top of the dry resin bed loosely cap and place the column back into the collection tube 6 Orient the spin column mark outward and centrifuge at 1 500 x g for 2 minutes 7 After centrifugation add 350 uL Buffer B to the conjugate solution located at the bottom of the collection tube and pipette up and down to mix Set aside on the bench for the time being G Q Spin Column Purification 40 min 15 minutes hands on 1 Pre wet a O Spin Filter see below by adding 200 uL Buffer B to the top of the filter unit and incubate for 2 minutes 14 mm gt lt filter unit a lt collection tube 2 Place the filter assembly into the centrifuge and orient the letter Q towards the center of the rotor spin at 2 000 x g for 4 minutes using an appropriate balance tube discard the flow through from collection tube and place the filter back into the empty collection tube 3 Load the antibody HRP conjugate 430 450 uL from the previous section section f step 7 to the top of the filter unit and allow it to incubate inside the filter for 2 minutes on the bench top 4 Place the oriented assembly in the centrifuge and spin at 2 000 x g for 4 minutes using an appropriate balance tube opposite the filter unit discard the flow through from the bottom collection tube and place the filter back into the empty collection tube Note a light brown coloring will appear on the top
7. May 2010 solulink HRP Antibody All in One Conjugation Kit User Manual Catalog No A 9002 001 Table of Contents Chapter 1 Introduction User Manual 5 Purpose of Manual 5 Intended Users 5 Customer Service and Technical Support 5 Chapter 2 Overview of Conjugation A Product Description 6 B All in One Technology 6 1 Conjugation Chemistry 6 2 Conjugate Purification 8 C All in One Conjugation Process Summary 9 D Materials Provided and Storage Conditions 10 E Additional Materials Required But Not Provided 10 Chapter 3 All in One Conjugation Protocol A IgG Sample Preparation 11 B Buffer Exchange IgG 11 C HyNic Modify IgG 12 D Buffer Exchange IgG 13 E Conjugate Formation 13 F Buffer Exchange Conjugate 14 G Q Spin Column Purification 14 H Buffer Exchange Conjugate 15 Chapter 4 Appendix A Polyclonal and Monoclonal IgG HRP Conjugates Some Examples 17 B Direct ELISA Assay Using an HRP IgG All in One Conjugate 18 C Bradford Protein Assay 19 D Using a NanoDrop to Measure Antibody Concentration 21 E HRP Absorption Spectra Unmodified Horseradish peroxidase 23 F 4FB modified HRP Absorption Spectra 23 G Bovine IgG HRP Conjugate Absorption Spectra All in One purified 24 H Concentration of Dilute Antibody Solutions 24 Troubleshooting Guide 26 J Component Stability on Storage 27 K References 28 The products offered here are for research use only Any commercial application will require a license fr
8. Monoclonal 5 ug 3 4FB modified HRP low loading barely visible 1 ug All in One Bovine lgG HRP conjugation reaction crude reaction 10 ug 5 All4n One spin column purified Anti FITC HRP conjugate crude reaction punfied 5 ug Figure 5 Coomassie stained 4 1296 SDS PAGE gels illustrating typical conjugation results Note horseradish peroxidase is a 44 kD highly glycosylated protein that migrates as a broad band when the protein sample is not heated 70 C before loading on the SDS PAGE gel 17 B Direct ELISA Assay Using an IgG HRP All in One Conjugate Figure 6 Direct ELISA curves generated using an HRP conjugate made with the All in One kit A mouse anti FITC monoclonal antibody was conjugated to HRP as described in the manual Antigen consisting of FITC labeled BSA FITC MSR 2 was coated on plates in a 2 fold dilution series 100 ul 500 250 125 62 5 31 25 15 625 7 8 3 90 and 1 95 ng ml using standard methods Immobilized antigen was then detected at 3 different conjugate concentrations 1 ug ml 0 5 ug ml 0 25 ug ml using TMB substrate 20 minutes 450 nm on a Molecular Devices plate reader 18 C Bradford Protein Assay Solulink highly recommends that whenever IgG is not limiting or its concentration source or quality are unknown that the sample be assayed for initial protein concentration using a Bradford protein assay prior to conjugation The starting quality and quantity of antibody is criti
9. a stable bis arylhydrazone bond HydraLinK is the only conjugation chemistry capable of efficiently converting nearly 100 of the antibody to its conjugate form This efficiency is made possible only because of the recent discovery that the aromatic compound aniline catalyzes this reaction 1 2 3 Aniline conjugation buffer also known and sold by its trade name as TurboLink M increases both the rate and efficiency of conjugate formation under mild reaction conditions resulting in reproducible and quantitative conversion of free antibody to conjugate Complete conversion of antibody to conjugate greatly simplifies downstream purification Purification consists of selectively binding the conjugate to a novel Q spin filter membrane that simply allows excess HRP to flow unbound through the membrane The spin filter provides high purity without sacrificing conjugate yield Conjugates made with All in One kit are compatible with such downstream applications as Westerns ELISAs or IHC Each kit provides sufficient reagents to perform two 2 conjugation reactions each yielding between 50 70 ug of high purity HRP antibody conjugate B All in One Technology 1 Conjugation Chemistry HydraLinK chemistry is based on the use of two complementary heterobifunctional linkers S HyNic and Sulfo S 4FB Figure 1 S HyNic Succinimidly 6 hydrazino nicotinamide is first used to modify and incorporate protected aromatic hydrazines HyNic groups into th
10. ature or refrigerated 2 8 C Flash Drive Room temperature E Additional Materials Required But Not Provided Bradford Protein Assay Reagents verification of initial IgG concentration Conventional UV VIS or NanoDrop Spectrophotometer optional Semi micro quartz cuvette 50 100 uL 1 cm path length For conventional UV VIS Spectrophotometer Calibrated pipettes P 2 or P 10 P 100 P 1000 and tips Variable speed centrifuge e g Eppendorf or MicroMax 1 5 ml microfuge tubes 10 Chapter 3 All in One Conjugation Protocol Important Before use remove the kit from the refrigerator and allow all kit components to warm up to ambient room temperature for at least 30 minutes A IgG Sample Preparation 10 minutes Antibodies come in two physical forms solids or liquids Individual samples can vary significantly in the amount of packaged IgG protein mass and or concentration mg ml We highly recommend that IgG concentrations be confirmed either by Bradford protein assay or A280 whenever possible The All in One M conjugation protocol requires antibody samples to be free of protein carriers such as BSA or gelatin before proceeding A 100 ug mass of antibody is required at the start of the procedure Depending on the initial form of your sample solid or liquid proceed as follows Antibody is in Solid Form e g lyophilized powder Resuspend lyophilized antibody 100 ug free of protein additives gelatin or BSA
11. cal to the success of the procedure A reference assay protocol is provided for measuring antibody or conjugate protein concentrations using Bradford protein reagents not provided in the kit Bradford Microtiter Plate Procedure Required Materials Bradford Reagent Bio Rad Hercules CA Cat 500 0006 96 well microtiter plate standard flat bottom PBS phosphate buffered saline P 200 and P 1000 pipettes Bovine IgG Antibody Standard 2 mg ml Pierce ThermoFisher Cat 23212 Molecular grade water Assay Protocol 1 Prepare 2 ml of a Bradford working solution by adding 400 ul dye reagent to 1600 ul molecular grade water 1 4 ratio 2 Prepare the following protein dilution standards and blank as follows Add 160 ul 2 mg ml bovine IgG standard to 240 ul PBS 0 8 mg ml standard Add 150 ul 0 8 mg ml standard to 50 ul PBS 0 6 mg ml standard Add 75 ul 0 6 mg ml standard to 25 ul PBS 0 4 mg ml standard Add 50 ul 0 4 mg ml standard to 50 ul PBS 0 2 mg ml standard Add 50 ul 0 2 mg ml standard to 50 ul PBS 0 1 mg ml standard Add 50 ul PBS buffer blank 3 Pipette 5 ul of each standard and blank along with duplicates of antibody Sample into separate microtiter wells 4 Add 100 ul of previously diluted dye reagent 1 4 to each well and mix thoroughly Always replace pipette tios between additions 5 Incubate at room temperature for 5 10 minutes but no more than 60 minutes 6 Measure absorbance at 595 nm on a S
12. e antibody via acylation of lysine residues In a similar fashion the second linker Sulfo S 4FB Sulfo N succinimidly 4 formylbenzamide is used by Solulink to form a pre activated high activity form of HRP called 4FB HRP provided for in the kit Incubation of the HyNic modified antibody with pre activated 4FB HRP in the presence of aniline as catalyst leads to rapid and efficient conversion of the antibody to conjugate through formation of stable bis arylhydrazone bonds Figure 2 Sulfo 5 4FB 0 C153 H14N4NaO4 M W 290 2 C2 Hag NOg4SNa M W 349 25 Figure 1 Structure of S HyNic and Sulfo S 4FB linkers used for conjugating HRP to antibody HyNic 4FB H o ae ae we NS H sot N N y HRP Aniline Catalyst IgG HRP Conjugate bis arylhydrazone bond Figure 2 Aniline catalyzed conjugation of HyNic modified antibody with pre activated 4FB HRP 2 Conjugate Purification The efficiency brought about by catalyzed hydrazone bond formation greatly simplifies conjugate purification Aniline s ability to increase both the rate and efficiency of conjugate formation under mild reaction conditions leads to nearly quantitative conversion of the free antibody to conjugate leaving behind only excess 4FB HRP After conjugation a novel Q spin filter is used to selectively bind the conjugate based on known biophysical properties of IgG 4 5 while allowing free HRP to be washed away In this manner purified conjugat
13. e is eluted from the filter membrane free of both residual antibody and HRP in high yield 50 70 ug X pi f IgG HRP conjugate free excess un conjugated 4FB modified HRP Bind conjugate to Q Spin Filter J E Re 2 m Bind conjugate to Q spin filter 6 9 Pig lt free HRP passes through Wash filter and discard wash J 2 Elute conjugate from filter J X X 100 pure IgG HRP conjugate Q Spin Filter Figure 3 O spin filter purification of HRP IgG conjugate C All in One Conjugation Process Summary Y m Buffer Exchange IgG JL co 3 min Grin gyo HyNic modify IgG 2 h O g i HyNic gt Excess S HyNic Buffer Exchange IgG 3 min HyNic linker B AFB HRP Conjugate IgG to HRP AF v 2h em Excess 4FB HRP Purify Conjugate 4 45 min O Spin Column Purified IgG HRP conjugate Figure 4 HRP Antibody All in One conjugation process D Materials Provided and Storage Conditions Components Amount Storage conditions S HyNic 2 X 100 ug Keep refrigerated within desiccated sealed aluminum pouch Spin Column Red cap Spin Column Yellow cap 2 Keep refrigerated 2 8 C O Spin filter Room temperature or refrigerated 2 8 C O collection tubes Room temperature or refrigerated 2 8 C DMF 0 5 mL Room temperature or refrigerated 2 8 C Diafiltration spin filters Room temperature or refrigerated 2 8 C Collection Tubes 16 Room temper
14. for 2 h at room temperature 12 D Buffer Exchange IgG 3 minutes 1 Five minutes before the end of the HyNic modification reaction section c step 3 prepare a spin column yellow cap by twisting off the bottom closure and loosening the yellow cap do not remove Place the spin column into a collection tube provided Mark the top of the yellow cap using an indelible pen to identify the sample Also place a vertical mark on the side of each spin column as shown on the next page Label the lid w sample ID Place pen mark on Side of spin column lt Collection tube Place the assembly into the centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor Use an appropriate balance tube opposite the assembly Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided Open the yellow cap load the now completed antibody HyNic modification reaction 25 35 uL to the top of the dry resin bed loosely cap and place the column back into the collection tube Orient the spin column mark outward and use an appropriate balance tube opposite the spin filter Centrifuge at 1 500 x g for 2 minutes After centrifugation transfer the solution 25 35 uL from the bottom of the collection tube to a 1 5 mL tube Label the
15. in 25 ul Buffer A to obtain a 4 mg mL solution If the antibody sample contains less than 100 ug per vial e g 50 ug resuspend the requisite number of vials equivalent to a 100 ug in 25 ul Buffer A to obtain a 4 mg mL solution Proceed to step B below Antibody is in Liquid Form e g PBS or TBS Buffer If the antibody sample is in liquid form at 4 mg ml simply transfer 25 ul to a labeled microfuge tube If the sample is in liquid form at a concentration greater than 4 mg ml transfer a volume equivalent to 100 ug antibody to a labeled microfuge tube and add Buffer A to obtain 4 mg ml solution If a sample is ata concentration less than 4 mg ml concentrate the sample to 25 uL and 4 mg mL using a diafiltration spin filter e g Amicon or VivaSpin 500 as described in the Appendix A concentration filter is provided for this purpose with the kit if necessary Proceed to step B below B Buffer Exchange IgG 3 minutes 1 Prepare a spin column red cap by twisting off the bottom closure and loosening the red cap do not remove Place the spin column into a collection tube provided 2 Mark the top of the red cap using an indelible pen to identify the sample and place a vertical mark on the side of each spin column as shown on the next page 11 Label lid w antibody ID Place pen mark on Side of spin column Collection tube Place the assembly into the centrifuge and orient the vertical mark on the spin column
16. lance tube opposite the spin filter Centrifuge for 2 minutes 5 000 x g Note never centrifuge for longer periods of time Open the filter unit and visually inspect the remaining volume If the volume remaining in the concentrator body is greater than 25 ul gently pipette the solution up and down to mix taking care not to touch or puncture the filter surface during this step Repeat steps 4 and 5 until the volume in the filter cup reaches the 25 ul mark Once the final volume reaches 25 ul do not pipette up and down to avoid sample loss Note if the volume goes lower than 25 ul at this stage add a small aliquot of Buffer A to bring the final volume to 25 ul Carefully transfer the concentrated IgG solution 25 ul to a new 1 5 ml microfuge tube and proceed with the rest of the procedure in Chapter III section B 20 Troubleshooting Guide Problem Poor conjugate yield Poor conjugate yield Poor HyNic modification Poor HyNic modification Possible Cause initial antibody concentration and volume were incorrect or unknown Starting antibody concentration and volume are incorrect or unknown presence of protein carrier e g BSA or gelatin is contaminating the antibody sample improper mixing of HyNic reaction components presences of amine contaminants improper storage of S HyNic reagent can lead to hydrolysis of this NHS ester 26 Recommended Action whenever possible verify the original sta
17. m 220 350 nm appears in the window Note for precious or limited samples the majority of the 2 ul aliquot can be recovered from the pedestal 9 Record the antibody concentration directly from the NanoDrop display window mg ml Alternately calculate the antibody concentration manually as illustrated on the following page 21 Example A mouse IgG sample at 1 mg ml in PBS 100 ul was scanned as described and its concentration confirmed using equation 1 below 230 240 250 280 270 280 280 300 310 320 330 340 35 SW eeselenath nm Figure 8 A mouse IgG sample 100 ul 2 1 mg ml in PBS pH 7 2 scanned on the NanoDrop as described in the text Sample Calculation Equation 1 Asa E1 value x 10 mg ml protein concentration mg ml E 1 mass extinction coefficient from Table 1 Example Mouse IgG 1 mg ml Fig 8 A280 reading from scan in Figure 8 1 34 Antibody E1 value Table 1 14 00 A280 E196 bovine IgG x 10 mg ml protein concentration mg ml 1 34 14 00 x 10 mg ml 0 96 mg ml Table 1 Mass extinction coefficients E196 used for calculating antibody concentrations The E196 is the Asa of a 10 mg ml solution in a 1 cm path 22 E HRP Absorption Spectrum Unmodified Horseradish peroxidase HRF Unmodified 0 66 mg ml Samples 11 Baseline 0 000 Al 280 Z Abs 0032 12 43 Abs2 0115 m O Cc iu O ci 2 1 E 0 0 4 I I I
18. nded Users The HRP Antibody All in One Kit is designed for users with minimal or no conjugation experience allowing them to prepare customized high purity ready to use HRP antibody conjugates in a single day Customer Service and Technical Support Additional technical information can be found at Telephone Email 1 888 625 0670 Toll Free Solulink Solulink com Fax Address 1 858 625 0770 Solulink The Conjugation Company 9853 Pacific Heights Blvd Ste H San Diego CA 92121 Chapter 2 Overview of Conjugation A Product Description Each HRP Antibody All in One Conjugation Kit provides all the necessary components to generate two 2 highly purified antibody HRP conjugates The kit requires the user to provide 100 ug of starting antibody for each conjugate The components of this unique kit feature a pre activated high activity horseradish peroxidase gt 250U mg as well as a novel Q spin filter to purify the conjugate in high yield Conjugates produced are free of both residual antibody and HRP thus providing maximum signal to noise in your assay Any suitably purified monoclonal or polyclonal mammalian antibody regardless of IgG subclass can be conjugated and purified within 5 hours 1 h hands on All in One conjugation kits are based on Solulink s patented HydraLink chemistry This chemistry relies on the specific reaction between an aromatic hydrazine HyNic and an aromatic aldehyde 4FB which leads to the formation of
19. of the Q filter membrane bound conjugate o Add 400 uL Buffer B to the filter unit orient and balance in the centrifuge and spin at 2 000 x g for 4 minutes discard the flow through from the bottom collection tube and place the filter back into the empty collection tube 6 Repeat step 5 two 2 additional times to complete the washing procedure 7 Remove the top filter unit from the collection tube and place it into a new collection tube provided 8 Add 100 uL Buffer C to the top of the brown colored filter membrane and incubate for 5 minutes on the bench top 9 Place the oriented assembly in the centrifuge and balance spin at 2 000 x g for 4 minutes add an additional 50 uL Buffer C to the top of the filter membrane and spin the oriented assembly for another 4 minutes at 2 000 x g A slightly brown colored conjugate solution 150 pL will now be visible at the bottom of the collection tube Set the collection tube aside on the bench H Buffer Exchange Conjugate 3 minutes 1 Prepare a spin column blue cap by twisting off the bottom closure and loosening the blue cap do not remove Place the spin column into a collection tube provided 2 Mark the top of the blue cap using an indelible pen to identify the sample Place a vertical mark on the side of each spin column as shown below 15 Label lid w conjugate ID Place pen mark on Side of spin column Collection tube Place the assembly into the cen
20. om Solulink 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 2009 Solulink The Conjugation Company 9853 Pacific Heights Blvd Suite H San Diego California 92121 All trademarks trade names service marks or logos referenced herein belong to their respective companies No license is granted or implied to any patents to technologies for which the end user applies our products For research purposes only Not for diagnostic use Safety Information WARNING CHEMICAL HAZARD Some chemicals used can be potentially hazardo
21. rting antibody concentration using a Bradford protein assay or NanoDrop to assure efficient conjugation concentrate or dilute the antibody sample to be conjugated into the reguired range 4 5 mg ml and 25 ul preservatives can interfere with the accuracy of a Bradford protein assay Remove all interfering preservatives such as thimerosal or proclin before performing a Bradford protein assay remove and purify away all protein carriers such as BSA or gelatin using affinity chromatography or other methods make sure to properly mix the antibody HyNic reaction mixture use a calibrated P 10 pipette to insure accuracy of small volumes remove all non protein amine contaminants such as glycine or Tris before modification keep and store S HyNic sealed in the aluminum pouch provided that contains dessicant initial antibody concentration was measure the initial too low or too high antibody concentration before proceeding Bradford or NanoDrop concentrate or dilute the antibody sample into the recommend range 4 5 mg ml and 25 ul before proceeding Low conjugate low spin column recovery volume use a properly calibrated and or antibody variable speed centrifuge recovery Follow recommended spin speed time Altered spin speeds can adversely compromise protein and or volume recovery J Component Stability on Storage Component Stability Storage Condition Unopened Ki Refrigerated 2
22. trifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor Use an appropriate balance tube opposite the spin filter Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided Remove the blue cap load 150 uL conjugate from the bottom of the Q spin filter collection tube section g step 9 to the top of the dry resin bed loosely recap and place the column back into the collection tube Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes After centrifugation transfer the buffer exchanged conjugate 150 uL from the bottom of the collection tube to a new 1 5 mL tube and label appropriately Measure the final protein concentration of the purified conjugate using either a Bradford or BCA protein assay see Appendix 16 Chapter 4 Appendix A Polyclonal amp Monoclonal IgG HRP Conjugates Some Examples Bovine IgG HRP Conjugate Mouse Anti FITC HRP Conjugate PanelA Panel B Panel A Protein M W Marker 2 HyNic modified Bovine IgG 5 ug 3 4FB modified HRP 5 ug 4 All in One Bovine lgG HRP conjugation reaction crude 10 ug 5 All in One spin column purified Bovine IgG HRP conjugate crude reaction purified 5 ug Panel B Protein M W Marker HyNic modified Mouse Anti FITC
23. uitable microtiter plate reader 7 A typical Bradford microtiter assay result from a commercial plate reader is illustrated below in Figure 7 19 S File Edit View Experiment Control Assays Group Window Help v C5 Bradford Protein Assay v U3 Plate 1 Setup E Template Reduction Display PZ Plate 1 H SO S J nl Wavelength Combination Lm1 Data Mode Absorbance Plate Blank Used Lm1 0 356 b Standards us 24 sft v Pl unknowns te Be fe Unknowns Wells OD Values Concentration Mean Conc Std Dev CV Dilution Adj Conc 1209 1299 0 000 00 10 1299 Sample 2 BAO 1020 1299 j3 cto X 0390 0 488 0 488 0000 oo 10 0 488 gt ff unkno din kau E4 ftal v Z Standard Curve it Fit Standard Curve Mean OD Value 0 4 0 5 Concentration v A Bx A O STD 1 Standards Concentration vs Mean OD Val 0 011 Figure 7 Print out from a Bradford microplate based protein assay 20 D Using a NanoDrop to Measure Antibody Concentration If an antibody sample is free of protein based carriers e g BSA gelatin or certain interfering preservatives such as thimerosal then a simple non destructive scan of the IgG sample on a NanoDrop spectrophotometer can be used to estimate antibody concentration saving the trouble of conducting a Bradford protein assay to confirm concentration To estimate antibody concentration
24. us and can cause injury or illness e Read and understand the Material Safety Data Sheets MSDS provided with this kit flash drive before you store handle or work with any chemicals or hazardous materials Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or clothing For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s clean up procedures as recommended in the MSDS e Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chapter 1 Introduction User Manual This manual provides instructions for using the HRP Antibody All in One Conjugation Kit This chapter contains the following sections Purpose ot Manual Intended Users Customer Service and Technical Support Purpose of Manual Each HRP Antibody All in One M Conjugation Kit provides all the necessary reagents and components to produce two 2 HRP antibody conjugates Use of the kit for each antibody results in e The modification of a user supplied antibody 100 ug with S HyNic e The conjugation of a HyNic modified antibody with 4FB HRP resulting in the formation of an HRP antibody conjugate e The isolation of highly purified HRP antibody conjugate using a rapid spin filter Q spin filter Inte
25. using a NanoDrop spectro photometer proceed as follows 1 Turn on the NanoDrop spectrophotometer and click on the NanoDrop icon to launch the software 2 Place a 2 ul drop of molecular grade water on the clean pedestal click OK 3 When the main menu appears select the Asa menu option Note do not use the UV VIS menu option on the NanoDrop to read an antibody sample 4 After the Asso menu appears click off the 340 nm normalization option using the mouse Note some instruments do not have this normalization feature in which case this step can be ignored 5 In the window labeled Sample Type select Other protein E1 option from the pull down menu Enter the appropriate E1 value Table 1 on the next page corresponding to your particular antibody sample type For example 14 00 for mouse IgG 6 Blank the NanoDrop spectrophotometer by placing a 2 ul drop of the appropriate sample buffer e g PBS and click on the Blank icon 7 Immediately re click the Measure icon to validate the baseline i e flat across the bandwidth Clean the pedestal and repeat if necessary until a flat baseline is obtained Note sometimes air bubbles can become trapped on the pedestal during sample loading and cause baseline offsets If necessary remove air bubbles and rescan to insure a proper baseline 8 Place a 2 ul volume of antibody solution on the clean pedestal and click the Measure icon Wait until the spectru

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