HRP-Antibody All-In-One Conjugation Kit Technical Manual
Contents
1. This chapter contains the following sections Purpose of Manual Intended Users Customer Service and Technical Support B Purpose of Manual Each HRP Antibody All in One 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 C Intended 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 D 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 o k Catalog A 9002 001 4 sol U N 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 co2. 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 o k Catalog A 9002 001 19 sol U nN 4 Add 100 ul of previously diluted dye reagent 1 4 to each well and mix thoroughly Always replace pipette tips 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 suitable microtiter plate reader 7 Atypical Bradford microtiter assay result from a commercial plate reader is illustrated below in Figure 7 o k Catalog A 9002 001 20 sol U nN File Edit View e Control mic Group Window Help SB ae ri C Bradford Protein Assay EU z Plate 1 Sutomix
3. After the Azs 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 jul 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 w k Catalog A 9002 001 2D sol U nN 8 Place a 2 ul volume of antibody solution on the clean pedestal and click the Measure icon Wait until the spectrum 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 Ex
4. 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 6 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 7 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 Ill section B M solulink Catalog A 9002 001 I Troubleshooting Guide Problem Poor conjugate yield Poor conjugate yield Poor HyNic modification Poor HyNic modification Catalog A 9002 001 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 28 Recommended Action whenever possible verify the original starting antibody concentration using a Bradford protein TM assay or NanoDrop to assure efficient conjugation concentrate or dilute the antibody sample to be conjugated into the required range 4 5 mg ml and 25 ul preservatives can interfere with the accur
5. n Conjugate Purification The efficiency of aniline 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 quantitative conversion of free antibody to conjugate The complete absence of free antibody at the end of the catalyzed reaction leaves only two components excess HRP and conjugate 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 flow through Purified conjugate can then be eluted from the filter membrane free of both residual antibody and HRP in high yield 50 70 ug Y o IgG HRP conjugate free excess un conjugated 4FB modified HRP Bind conjugate to Q Spin Filter 4 Bind conjugate to Q spin filter 00 free HRP passes through Q Spin Filter Wash filter and discard wash 4 Elute conjugate from filter 4 TX 100 pure IgG HRP conjugate Figure 3 Q spin filter purification of HRP lgG conjugate solulink Catalog A 9002 001 7 C Il in One M Conjugation Process Summary Buffer Exchange IgG 3 min HyNic modify IgG 2 h Buffer Exchange IgG 3 min Conjugate IgG to HRP 2 h Purify Conjugate 45 min Y o S HyNic linker HyNic Y Excess S HyNic 4FB k 4FB HRP K 4 Excess 4FB HRP Q Spin Column Purified
6. IgG HRP conjugate Figure 4 HRP Antibody All in One conjugation process Catalog A 9002 001 solulink D Materials Provided and Storage Conditions Components Amount Storage conditions S HyNic 2x100 ug Keep refrigerated within desiccated sealed aluminum pouch Q Spin Filters 2 Room temperature or refrigerated 2 8 C oele NN 2 8 C 2 8 C Room temperature or refrigerated 30k 2 8 C 2 8 C E Additional Materials Required But Not Provided 4 Room temperature or refrigerated 6 Bradford Protein Assay Reagents verification of initial IgG concentration Conventional UV VIS or NanoDrop M 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 Catalog A 9002 001 9 sol U nN Chapter 3 All in One Conjugation Protocol Note Before using the kit remove from refrigerated storage and allow components to warm up to ambient or 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 whene
7. fo 0 10 Abs 1 0040 1 mm Absorbance N A Abs 2 0 085 Ey l l l I l l l l I l 1 eel 240 260 280 300 320 340 360 300 400 420 440 460 dal 500 520 550 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 G Bovine IgG HRP Conjugate Absorption Spectrum All in One Purified ampie IL Mouse HRP IgG All In One Moniunato a om co con Sample 6 oe Mm om Baseline 0 000 mo AL 280 nm om Abs 1 0 195 w Oo fa wu 9 4 z si ao A2 403 i nm 0 05 Abs 2 0 101 0 00 l l l l l l l l l 220 240 260 260 300 320 340 360 350 400 420 440 460 450 500 Wavelength nm Figure 11 NanoDrop absorption spectrum of All in One IgG HRP conjugate 220 550 nm 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 requires 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 0 25 to 1 5 mg ml In these solulink Catalog A 9002 001 25 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
8. 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 of the Q filter membrane bound conjugate 5 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 ano
9. 001 11 sol U nN 7 Transfer the buffer exchanged IgG solution 25 30 uL from the bottom of the collection tube to a new 1 5 mL tube and label appropriately C HyNic Modify IgG 2 hours 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 2 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 3 Incubate the reaction for 2 h at room temperature 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 2 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 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 assembly w k Catalog A 9002 001 12 sol U nN 4 Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube T
10. A Assay Using an IgG HRP All in One Conjugate Direct ELISA Standard Curves Lug ml 0 5 ug ml 0 25 ug ml Z DUS GO SKK PO OI 1 10 100 1000 Antigen Concentration ng ml 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 o k Catalog A 9002 001 18 sol U nN 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 critical 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
11. ARD Some chemicals used can be potentially hazardous and can cause injury or illness e Read and understand the Material Safety Data Sheets MSDS available at Solulink com before you store handle or work with any chemicals or hazardous materials e 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 e 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 o k Catalog A 9002 001 2 Sol U N Table of Contents Chapter ato 7 esneari ee ee eee ee ee 4 BNSC AS aa atasan 4 Be Purbo MANA en na AR Dn 4 Cs P ended Uce ea me ne MO eee ee ee Ee nen eer ere ene aeeeer 4 D Customer Service and Technical Support ooooooWoo oo 4 Chapter 2 Overview of Conjugation oooo naek 5 A Produce Des OM an on toa aman ena 5 Bs Aeon ee asa 5 C All in One Conjugation Process SUMMATY oooooooooo Woo Woman anna 8 D Materials Provided and Storage Conditions ooooooo anna 9 E Additional Materials Required But Not Provided coooooo Woo 9 Chapter 3 All in One Conjugation Protocol ooooooooo
12. Once Calibrate On Column Priority Wavelength Combination Lim Data Mode Absorbance Plate Blank Used Lint 0 356 FTI Standards Lat E4 e Unknowns ua EJ Fta Unknowns OD Values Std Dev Ae ato Adj Cons 1 020 1 299 1 200 o 000 oof 4 0 1 299 2 3 jmo zoo oss oa e 0000 oo 1o o4 b FER unkno ciiny H Be e 7 Z Standard Cuveits Fit Standard Curve Mean OD Value O 4 0 5 Concentration y Ox 4 0 STO 1 Standards Concentration vs Mean OD Wal Figure 7 Print out from a Bradford microplate based protein assay solulink Catalog A 9002 001 21 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 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 a2 ul drop of molecular grade water on the clean pedestal click OK 3 When the main menu appears select the A239 menu option Note do not use the UV VIS menu option on the NanoDrop to read an antibody sample 4
13. Version 5 27 10 solulink HRP Antibody All In One Conjugation Kit Technical Manual Catalog A 9002 001 Note This protocol and any documents linked below can be downloaded from the appropriate category in the Solulink Library at http www solulink com library solulink Catalog A 9002 001 1 Disclaimer The products offered here are for research use only Any commercial application will require a license from 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 Safety Information WARNING CHEMICAL HAZ
14. 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 ug 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 Concentrator body lang F 5 b d Filtrate tube Figure 12 Diafiltration spin filter used for concentrating dilute antibody samples prior to the start of All in One conjugation protocol 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 1 Open the lid of a diafiltration spin filter device 2 Transfer 500 ul or less of dilute protein solution equivalent to 125 ug antibody to the center of the filter cup 3 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 balance tube opposite the spin filter 4 Centrifuge for 2 minutes 5 000 x g w k Catalog A 9002 001 26 sol U nN 5 Open the filter unit and visually inspect the remaining volume If the volume remaining
15. acy 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 solulink initial antibody concentration was too low or too high Low conjugate and or low spin column recovery volume antibody recovery J Component Stability on Storage contains dessicant measure the initial 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 use a properly calibrated variable speed centrifuge Follow recommended spin speed time Altered spin speeds can adversely compromise protein and or volume recovery Component Stability Storage Condition Unopened Kit S HyNic 24 h after re suspending S HyNic in DMF HRP Antibody 9 month Conjugate All other kit components Catalog A 9002 001 29 Refrigerated 2 8 C Keep in sealed aluminum pouch provided 2 8 C Room temperature Refrigerated 2 8 C i
16. ample A mouse IgG sample at 1 mg ml in PBS 100 ul was scanned as described and its concentration confirmed using equation 1 below 240 250 260 270 280 Wavelength nm Figure 8 A mouse IgG sample 100 ul 1 mg ml in PBS pH 7 2 scanned on the NanoDrop as described in the text Equation 1 A 9 E1 value x 10 mg ml protein concentration mg ml E1 mass extinction coefficient from Table 1 Example Mouse IgG 1 mg ml Fig 8 Asso reading from scan in Figure 8 1 34 Antibody E1 value Table 1 14 00 Asso E1 bovine IgG x 10 mg ml protein concentration mg ml 1 34 14 00 x 10 mg ml 0 96 mg ml solulink Catalog A 9002 001 23 Table 1 Mass extinction coefficients E1 used for calculating antibody concentrations The E1 is the A gp of a 10 mg ml solution in a 1 cm path E HRP Absorption Spectrum Unmodified Horseradish peroxidase 0 35 HRP Unmodified 0 66 mg ml 0 30 a PEN Sample 1 tt fo il Abs 1 0 032 1 mm Absorbance 0 10 O Ao N oles 0 05 0 01 l l I l I l l I l l l l l I l l 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 550 Wavelength hm 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 va solulink Catalog A 9002 001 F 4FB modified HRP Absorption Spectrum a 4FE HRP 0 66 mg ml TP li nan Ni o E Wi
17. 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 w k Catalog A 9002 001 10 sol U nN Label lid w antibody ID Place pen mark on Side of spin column lt 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 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 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 6 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 Note Rotor speed must be set to 1500 x g RCF and not 1500 x rpm 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 o k Catalog A 9002
18. compatible with all downstream applications such 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 Conjugation Chemistry HydraLink M chemistry is based on the use of two complementary heterobifunctional linkers S HyNic and Sulfo S 4FB Figure 1 S HyNic Succinimidyl 6 hydrazino nicotinamide is first used to modify and incorporate protected aromatic hydrazines HyNic groups into the antibody via acylation of lysine residues In a similar fashion a second linker Sulfo S 4FB Sulfo N succinimidyl 4 formylbenzamide is used by Solulink to provide a pre activated high activity HRP called 4FB HRP included Incubation of HyNic modified antibody with pre activated 4FB HRP in the presence of aniline catalyst leads to rapid and efficient conversion of the antibody to conjugate through formation of stable bis arylhydrazone bonds Figure 2 o k Catalog A 9002 001 5 Sol U n S HyNic Sulfo S 4FB O Cys Hia Ma NaDa MIA 290 2 Caz HaNOaSMat MIW 349 25 Figure 1 Structure of S HyNic and Sulfo S 4FB linkers used for conjugating HRP to antibody HyNic mol a Jbg i Aniline Catalyst IgG HRP Conjugate bis arylhydrazone bond Figure 2 Aniline catalyzed conjugation of HyNic modified antibody with pre activated 4FB HRP w k Catalog A 9002 001 6 sol U
19. f each spin column as shown below w k Catalog A 9002 001 13 sol U nN Label lid w conjugate ID a pe Place pen mark on side of spin column lt amp 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 uL 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 1 Pre wet a Q Spin Filter refer to next page by adding 200 uL Buffer B to the top of the filter unit and incubate for 2 minutes nga gt filter unit ae eee Qa collection tube o k Catalog A 9002 001 14 sol U nN 2 Place the filter assembly into the centrifuge and orient the letter Q towards the center of the rotor spin at 2
20. fer the buffer exchanged conjugate 150 uL from the bottom of the collection tube to a new 1 5 mL tube and label appropriately w k Catalog A 9002 001 16 sol U nN 8 Measure the final protein concentration of the purified conjugate using either a Bradford or BCA protein assay see Appendix Chapter 4 Appendix A Polyclonal amp Monoclonal IgG HRP Conjugates Some Examples Bovine IgG HRP Conjugate Mouse Anti FITC HRP Conjugate t 2 3 4 5 1 1 La on a a rt si f l q Fiora dei a Ai a TA i _ os hg E e 1 Pa Panel A Panel A Protein M W Marker HyNic modified Bovine IgG 5 yg 4FB modified HRP 5 pg All in One Bovine lgG HRP conjugation reaction crude 10 yg Allin One spin column punfied Bovine lgG HRP conjugate crude reaction purified 5 yg Panel B Protein M W Marker HyNic modified Mouse Anti FITC Monoclonal 5 pq 4FB modified HRP low loading barely visible 1 ug Allin One Bovine IgG HRP conjugation reaction crude reaction 10 yg All in One spin column purified Anti FITC HRP conjugate crude reaction punfied 5 pq Figure 5 Coomassie stained 4 12 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 an SDS PAGE gel solulink Catalog A 9002 001 17 B Direct ELIS
21. he column matrix will appear white in color Place the column back into a new empty collection tube provided 5 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 6 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 7 After centrifugation transfer the solution 25 35 uL from the bottom of the collection tube to a 1 5 mL tube Label the sample appropriately e g HyNic IgG E Conjugate Formation 2 hours 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 2 Transfer 50 ul 4FB modified HRP to the tube containing HyNic modified antibody 25 35 uL pipette up and down to mix 3 Incubate the reaction for 2 h at room temperature to form the conjugate 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 o
22. n final conjugate solution Refrigerated 2 8 C 50 glycerol Refrigerated 2 8 C solulink K References 1 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 Lim S Manusu H P Gooley A A Williams K L Rylatt D B 1998 Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis Journal of Chromatography A Vol 827 Issue 2 11 Pages 329 335 Chiodi F Sid n A Osby E 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 o k Catalog A 9002 001 30 sol U nN
23. njugate 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 M conjugation kits are based on Solulink s proven HydraLink chemistry This chemistry involves the reaction of an aromatic hydrazine with an aromatic aldehyde to form a stable hydrazone bond HydraLinK conjugation is so efficient that it converts 100 of the antibody to the conjugate form This linking efficiency is made possible because of the recent discovery that small quantities of aniline catalyze hydrazone bond formation between the two functional groups 1 2 3 Aniline increases both the rate and efficiency of conjugate formation under mild reaction conditions leading to quantitative conversion of free antibody to HRP 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 allows excess HRP to flow through unbound The spin filter provides high purity without sacrificing conjugate yield Conjugates made with All in One kit are
24. oooooo oo 10 A IgG Sample Preparation 10 minutes o oooooo ooh 10 Bs Burner Exchange gG ie ten Kanaan uno 10 CO Ae Mon yV eG ZNO an an en banaai 12 D B ffer Exchange lgG 3 MINUTES na stauesaadenseesiaatonennaramee 12 E Conjugate Formation 2 ho EE 13 F Buffer Exchange Conjugate 3 minutes oo Rana 13 G Q Spin Column Purification 40 minutes 14 H Buffer Exchange Conjugate 3 minutes ooooooooooooo nanah 16 Chapter Ape A eee 17 A Polyclonal amp Monoclonal IgG HRP Conjugates Some Examples oo Woo 17 B Direct ELISA Assay Using an IgG HRP All in One Conjugate oooooooooooooooomm 18 Ce Brodlord Protein ASS ay ta 19 D Using a NanoDrop to Measure Antibody Concentration oooooco oWo oo ooo oooooo 22 E HRP Absorption Spectrum Unmodified Horseradish peroxidase ooooWoo 24 F AFB modified HRP Absorption Spectrum oooooo Woman 25 G Bovine IgG HRP Conjugate Absorption Spectrum All in One Purified o ooooooooooomo 25 H Concentration of Dilute Antibody Solutions oooooo Won 25 i Troubleshooting Guide ooooooo on 28 J Component Stability on Storage oooooooo anna 29 ke RIGEN E aan an NN E na PAN NA DA PA AN DE NA NN PA 30 o k Catalog A 9002 001 3 Sol U n Chapter 1 Introduction A User Manual This manual provides instructions for using the HRP Antibody All in One Conjugation Kit
25. ther 4 minutes at 2 000 x g A slightly brown colored conjugate solution 150 uL will now be visible at the bottom of the collection tube Set the collection tube aside on the bench w k Catalog A 9002 001 15 sol U nN 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 on the next page Label lid w conjugate ID Place pen mark on Side of spin column lt 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 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 6 Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes 7 After centrifugation trans
26. ver possible The All in One 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 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 at a 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 in the kit for this purpose 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
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