10012445

Transcript

1 His-Tag Detection ELISA Kit Item No. 10012445 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd · Ann Arbor, MI · USA

2 TABLE OF CONTENTS GENERAL INFORMATION 3 GENERAL INFORMATION Materials Supplied Materials Supplied Safety Data 4 4 Precautions Item Number Item 96 wells If You Have Problems 4 Quantity/Size Storage and Stability 5 400242 1 vial/100 dtn His ELISA Monoclonal Antibody Materials Needed but Not Supplied 5 1 vial/100 dtn His-AP Tracer 400240 INTRODUCTION 6 Background 1 vial His-Protein ELISA Standard 400247 6 About This Assay TBS Assay Buffer Concentrate (10X) 400084 2 vials/10 ml 7 Principle of the Assay 411007 AP Wash Buffer Concentrate (150X) 1 vial/5 ml 9 Definition of Key Terms 1 plate Goat Anti-Mouse IgG Coated Plate 400008/400009 Buffer Preparation 10 PRE-ASSAY PREPARATION 400012 96-Well Cover Sheet 1 cover Sample Preparation 11 2 vials/12 ml NPP Substrate Solution 400089 p ASSAY PROTOCOL 12 Preparation of Assay-Specific Reagents 15 Plate Set Up If any of the items listed above are damaged or missing, please contact our 16 Performing the Assay - Customer Service department at (800) 364-9897 or (734) 971-3335. We cannot Non-Imidazole accept any returns without prior authorization. ANALYSIS 19 Calculations WARNING: THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR Performance Characteristics 21 HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE. ! 23 Appendix RESOURCES 28 Troubleshooting 29 References Plate Template 30 31 Notes Warranty and Limitation of Remedy 31 GENERAL INFORMATION 3

3 Safety Data Storage and Stability This kit will perform as specified if stored at -20 ° This material should be considered hazardous until further information becomes C and used before the expiration available. Do not ingest, inhale, get in eyes, on skin, or on clothing. Wash date indicated on the outside of the box. thoroughly after handling. Before use, the user Safety complete review the must Data Sheet, which has been sent via email to your institution. Materials Needed But Not Supplied A plate reader capable of measuring absorbance between 405-420 nm. 1. Precautions Adjustable pipettes and a repeating pipettor. 2. Please read these instructions carefully before beginning this assay. A source of ‘UltraPure’ water. Water used to prepare all ELISA reagents and 3. This kit may not perform as described if any reagent or procedure is replaced or buffers must be deionized and free of trace organic contaminants (‘UltraPure’). modified. Use activated carbon filter cartridges or other organic scavengers. Glass distilled water (even if double distilled), HPLC-grade water, and sterile water If You Have Problems (for injections) are not adequate for ELISA. NOTE: UltraPure water is available for purchase from Cayman (Item No. 400000). Technical Service Contact Information Buffer Preparation (see pages Materials used for Sample Preparation and 4. 888-526-5351 (USA and Canada only) or 734-975-3888 Phone: 10 and 11, respectively). Fax: 734-971-3641 [email protected] Email: M-F 8:00 AM to 5:30 PM EST Hours: In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box). GENERAL INFORMATION GENERAL INFORMATION 5 4

4 Principle of the Assay INTRODUCTION This assay is based on the competition between free His-protein and a 6X-His Background Tracer (6X-His linked to alkaline phosphatase (AP)) for a limited number of His- specific monoclonal antibody binding sites. The concentration of the 6X-His Tracer Recombinant protein expression is a valuable tool in the production of large is held constant while the concentration of free His-protein (standard or sample) amounts of protein for both functional and structural studies. To facilitate varies. Thus, the amount of Tracer that is able to bind to the monoclonal antibody purification and detection, recombinant proteins are often labeled with affinity will be inversely proportional to the concentration of free His-protein in the well. tags, such as hexahistidine (6X-His), GST, and FLAG. 6X-His tags are often This monoclonal antibody-His (either free or tracer) complex binds to the goat the tag of choice due to their small size, less potential to interfere in protein anti-mouse IgG that has been previously attached to the well. The plate is washed 2+ ions. His-tagged proteins folding, weak immunogenicity, and high affinity for Ni p NPP is added to the well. The product to remove any unbound reagents and then 2+ expressed in bacteria or baculovirus/insect cells can be easily purified by Ni - of this enzymatic reaction has a distinct yellow color and absorbs strongly at 1 Cell lysates and samples at different stages of purification resin chromatography. 412 nm. The intensity of this color, determined spectrophotometrically, is are generally analyzed by SDS-PAGE to determine the expression of His-proteins, proportional to the amount of Tracer bound to the well, which is inversely which can add several days to the purification and analysis protocol. A semi- proportional to the amount of free His-protein present in the well during the quantitative screening assay would provide the ability to rapidly screen for His- incubation; or tagged proteins at each stage of expression and purification, permitting the user Absorbance ∝ [Bound His Tracer] 1/[His-protein] ∝ to determine if there is sufficient protein expression to continue with purification and to monitor loss or enrichment at each stage. A schematic of this process is shown in Figure 1 on page 8. About This Assay Cayman’s His-Tag Detection ELISA Kit is a competitive assay designed for the rapid, semi-quantitative screening of cell lysates and affinity column fractions for His-tagged proteins. It can be used as a substitute for SDS-PAGE to expedite the screening of affinity column fractions within a few hours. INTRODUCTION INTRODUCTION 7 6

5 Definition of Key Terms p NPP Reagent. The blank absorbance background absorbance caused by Blank: all the other wells, should be subtracted from the absorbance readings of including NSB wells. use Ig Mo Po = Goat clonal G An⑱- ly Plates ar e pr e-coated Incubate , acer with tr 1. locking pr = B otein s non-immunological binding of the tracer to the well. NSB (Non-Specific Binding): clonal with goat poly an⑱body , and either an⑱-mouse I gG and sample. d or standar race (t = A X- ed to 6 link His P r) Even in the absence of specific antibody a very small amount of tracer still binds block ed with a pr oprietary rmula⑱on of pr oteins. fo to the well; the NSB is a measure of this low binding. Do not forget to subtract = S to His pecific an⑱body the Blank absorbance values. r ee His-P ro tein = F B (Maximum Binding): maximum amount of the tracer that the antibody can 0 bind in the absence of free analyte. ove all Wa sh to r em well with Develop the 2. 3. ratio of the absorbance of a particular %B/B (%Bound/Maximum Bound): 0 re unbound . agents solu⑱on p NPP . sample or standard well to that of the maximum binding (B ) well. 0 Figure 1. Schematic of the ELISA values concentration of a series of versus a plot of the %B/B Standard Curve: 0 wells containing various known amounts of analyte. Dtn: determination, where one dtn is the amount of reagent used per well. INTRODUCTION INTRODUCTION 9 8

6 Sample Preparation PRE-ASSAY PREPARATION Crude cell lysates contain materials that interfere in the assay and give false NOTE: Water used to prepare all ELISA reagents and buffers must be deionized positives or erroneously high levels of His-tagged protein. It is recommended and free of trace organic contaminants (‘UltraPure’). Use activated carbon filter that cell lysates be cleared by high speed centrifugation before assaying. cartridges or other organic scavengers. Glass distilled water (even if double distilled), NOTE: Due to the interference of denaturing reagents, this assay is not suitable for HPLC-grade water, and sterile water (for injections) are not adequate for ELISA. require denaturing to expose the His-tag. proteins that UltraPure water may be purchased from Cayman (Item No. 400000). Buffers and reagents that have been tested and demonstrated to be compatible Buffer Preparation with the assay: Store all diluted buffers at 4°C; they will be stable for about two months. Reagents 1. TBS Assay Buffer Preparation Dilute the contents of one vial of TBS Assay Buffer Concentrate (10X) PBS (phosphate buffered saline) Compatible (Item No. 400084) with 90 ml of UltraPure water. Be certain to rinse the vial to remove any salts that may have precipitated. NOTE: It is normal for Buffers and PBS (1.0% NP-40) the concentrated buffer to contain crystalline salts after thawing. These will Reagents completely dissolve upon dilution with water. TBS (Tris buffered saline) 2. AP Wash Buffer Preparation 50 mM potassium phosphate, 500 mM sodium chloride, pH 7.2 Dilute the contents of the vial of AP Wash Buffer Concentrate (150X) (Item No. 411007) to a total volume of 750 ml with UltraPure water. Smaller 50 mM potassium phosphate, 500 mM sodium chloride (1.0% NP-40) volumes of Wash Buffer can be prepared by diluting the Wash Buffer Concentrate 1:150. B-PER Protein extraction reagent Imidazole (<300 mM) Urea (>0.5 M) Incompatible Reagents Guanidine-HCl (>0.5 M) Imidazole (>300 mM) PRE-ASSAY PREPARATION PRE-ASSAY PREPARATION 11 10

7 500 μl 500 μl 500 μl 500 μl 500 μl 60 μl ASSAY PROTOCOL Preparation of Assay-Specific Reagents S2 S4 S1 S5 S6 S3 His-Protein ELISA Standard This kit contains one His-Protein ELISA Standard (Item No. 400247). As this is a rapid screening assay, and the immunoreactivity of different His-tagged proteins Final 2 is highly variable, the standard is not designed to be used as a quantitative tool, but provide a means of monitoring protein gain or loss during the various 500 μl 500 μl 500 μl 500 μl 500 μl 940 μl TBS TBS TBS TBS TBS TBS purification steps. Assa y y Assa Assa y y Assa y Assa Assa y er Bu er Bu er Bu Bu er er Bu er Bu NOTE: A standard curve is NOT recommended for screening column fractions eluted 0.187 0.093 3 0.375 0.75 1.5 50 μg/ml in imidazole (see page 20). μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml Bulk Standar d Reconstitute the His-Protein Standard with 400 μl TBS Assay Buffer. The Figure 2. Preparation of the His-Protein standard concentration of this solution (the bulk standard) will be 50 μg/ml. Store this solution at 4°C; it will be stable for at least two weeks. For longer storage, we recommend the bulk standard be aliquoted and stored at -20°C or lower. Avoid repeated freeze/thaw cycles. To prepare the standard for use in ELISA: Obtain six clean test tubes and number them #1 through #6. Aliquot 940 μl TBS Assay Buffer to tube #1 and 500 μl TBS Assay Buffer to tubes #2-6. Transfer 60 μl of the standard to tube #1 and mix thoroughly. Serially dilute the standard by removing 500 μl from tube #1 and placing in tube #2; mix thoroughly. Next, remove 500 μl from tube #2 and place it into tube #3; mix thoroughly. Repeat this process for tubes #4-6. ASSAY PROTOCOL ASSAY PROTOCOL 13 12

8 His-AP (Alkaline Phosphatase) Tracer Plate Set Up Reconstitute the His-AP Tracer (Item No. 400240) with 6 ml TBS Assay Buffer. The 96-well plate(s) included with this kit is supplied ready to use. It is not and use within two (do not freeze!) Store the reconstituted His-AP Tracer at 4°C necessary to rinse the plate(s) prior to adding the reagents. NOTE: If you do not weeks. A 20% surplus of tracer has been included to account for any incidental need to use all the strips at once, place the unused strips back in the plate packet and losses. store at 4°C. Be sure the packet is sealed with the desiccant inside. There is no specific pattern for using the wells on the plate. A suggested plate His ELISA Monoclonal Antibody format is shown in Figure 3, below. Each plate or set of strips should contain a Reconstitute the His ELISA Monoclonal Antibody (Item No. 400242) with 6 ml minimum of two blanks (Blk), two non-specific binding wells (NSB), two maximum TBS Assay Buffer. Store the reconstituted His ELISA Monoclonal Antibody at binding wells (B ), and a six point standard curve run in duplicate. NOTE: Each 0 4°C. It will be stable for at least four weeks. A 20% surplus of antibody has been assay must contain this minimum configuration in order to ensure accurate and included to account for any incidental losses. Each sample should be assayed at two dilutions and each reproducible results. dilution should be assayed in duplicate. For statistical purposes, we recommend assaying samples in triplicate. We suggest you record the contents of each well on the template sheet provided (see page 30). 12345 678 9101112 A B 8 B 8 S1 24 32 32 16 24 16 S1 0 0 B 1 9 1 S2 9 S2 25 33 17 33 25 17 Blk - Blank 2 10 10 2 26 34 18 34 S3 26 18 S3 C NSB - Non-Specific Binding B - Maximum Binding 11 3 3 11 27 35 19 35 27 19 S4 D S4 0 S1-S6 - Standards 1-6 4 12 S5 12 28 36 20 36 E 28 20 4 S5 1-39 - Samples 5 29 37 21 37 29 21 13 S6 13 S6 F 5 14 14 6 30 38 22 38 NSB 30 NSB 22 6 G 7 7 15 15 31 39 23 39 31 23 Blk Blk H Figure 3. Sample plate format ASSAY PROTOCOL ASSAY PROTOCOL 15 14

9 Performing the Assay (Non-Imidazole) Well TBS Assay Standard/ Sample AP Tracer His ELISA Monoclonal Antibody Buffer Instructions for Imidazole fractions (<300 mM imidazole) can be found on pages 23-27. - Blk - - - Pipetting Hints μ 100 NSB 50 l - μ l - • Use different tips to pipette each reagent. μ μ 50 - l μ 50 l 50 l B 0 • Before pipetting each reagent, equilibrate the pipette tip in that , slowly fill the tip and gently expel the contents, repeat i.e. reagent ( Std/Sample l 50 μ - l 50 μ l 50 μ several times). Do not expose the pipette tip to the reagent(s) already in the well. • Table 1. Pipetting summary Addition of the Reagents 1. TBS Assay Buffer Add 100 μl TBS Assay Buffer to NSB wells. Add 50 μl TBS Assay Buffer to B wells. 0 2. His-Protein ELISA Standard Add 50 μl from tube #6 to both of the lowest standard wells (S6). Add 50 μl from tube #5 to each of the next two standard wells (S5). Continue with this procedure until all the standards are aliquoted. The same pipette tip should be used to aliquot all the standards. Before pipetting each standard, be sure to equilibrate the pipette tip in that standard. 3. Samples Add 50 μl of sample per well. Each sample should be assayed at a minimum of two dilutions. Each dilution should be assayed in duplicate (triplicate recommended). 4. His-AP Tracer Add 50 μl to each well except the Blk wells. 5. His ELISA Monoclonal Antibody except the NSB and the Blk wells. Add 50 μl to each well ASSAY PROTOCOL ASSAY PROTOCOL 17 16

10 Incubation of the Plate ANALYSIS Cover each plate with plastic film (Item No. 400012) and incubate for 90 minutes Many plate readers come with data reduction software that plots data at room temperature on an orbital shaker. automatically. Alternatively a spreadsheet program can be used. The data should log concentration using a four-parameter versus be plotted as either %B/B 0 Development of the Plate NOTE: log concentration using a linear fit. versus logistic fit or as logit B/B 0 1. Empty the wells and rinse four times with Wash Buffer. Each well should Cayman Chemical has a computer spreadsheet available for data analysis. Please be completely filled with Wash Buffer during each wash. Invert the plate contact Technical Service or visit our website (www.caymanchem.com/analysis/elisa) between wash steps to empty the fluid from the wells. After the last wash, to obtain a free copy of this convenient data analysis tool. gently tap the inverted plate on absorbent paper to remove the residual Wash Buffer. Calculations Add 200 μl of 2. p NPP Substrate Solution to each well of the plate. Preparation of the Data 3. Cover the plate with plastic film and incubate for approximately 60 minutes at room temperature. The assay typically develops in 30-60 minutes. The following procedure is recommended for preparation of the data prior to graphical analysis. Reading the Plate NOTE: If the plate reader has not subtracted the absorbance readings of the blank Wipe the bottom of the plate with a clean tissue to remove fingerprints, dirt, 1. wells from the absorbance readings of the rest of the plate, be sure to do that now. etc. Average the absorbance readings from the NSB wells. 1. 2. Read the plate at a wavelength between 405 and 420 nm. The absorbance wells. 2. Average the absorbance readings from the B 0 wells have reached a minimum of may be checked periodically until the B 0 Subtract the NSB average from the B 3. or average. This is the corrected B 0.3 A.U. (blank subtracted). The plate should be read when the absorbance 0 0 corrected maximum binding. wells in the range of 0.3-1.5 A.U. (blank subtracted). of the B 0 4. Calculate the B/B (Sample or Standard Bound/Maximum Bound) for the 0 remaining wells. To do this, subtract the average NSB absorbance from the S1 absorbance and divide by the corrected B (from Step 3). Repeat for 0 S2-S8 and all sample wells. (To obtain %B/B for a logistic four-parameter 0 fit, multiply these values by 100.) ANALYSIS ASSAY PROTOCOL 18 19

11 Plot the Standard Curve Performance Characteristics for standards S1-S6 versus His-Protein concentration using linear (y) Plot %B/B 0 Due to variability in immunodetection of the His-tag on His-tagged proteins, the and log (x) axes and perform a 4-parameter logistic fit. sensitivity of this assay will vary depending on the protein being analyzed. The Alternative Plot - The data can also be lineraized using a logit transformation. purified His-tagged standard provided typically show a sensitivity of 100 ng/ml. in this NOTE: Do not use %B/B The equation for this conversion is shown below. If the tertiary structure of the experimental protein partially occludes the His-tag, 0 calculation. the sensitivity of the assay may be reduced. /(1 - B/B ) = ln [B/B )] logit (B/B 0 0 0 10 0 Plot the data as logit (B/B versus log concentrations and perform a linear ) 0 regression fit. 80 Determine the Sample Concentration ) value for each sample. Determine the concentration (or %B/B Calculate the B/B 0 0 60 of each sample by using the equation obtained from the standard curve plot. 0 NOTE: Remember to account for any dilution or concentration of the sample prior to values greater than 80% or less than Samples with %B/B the addition to the well. %B/B 0 40 20% should be re-assayed as they generally fall out of the linear range of the standard curve. A 20% or greater disparity between the apparent concentration of two different dilutions of the same sample indicates interference in the assay. 20 0 1 10 0.1 His-Pro tein (μg/ml) Figure 4. Typical standard curve ANALYSIS ANALYSIS 21 20

12 50 RESOURCES Appendix 40 (<300 mM Imidazole) Performing the Assay - Imidazole fractions 30 Plate Set Up μg/ml Due to varying degrees of interference of imidazole in the assay, control wells 20 containing imidazole buffer alone MUST be included. For example, if proteins are eluted with 100 mM, 200 mM, and 300 mM imidazole from a column, control wells containing equivalent concentrations of imidazole must also be included. A 10 standard curve is NOT recommended for screening column fractions. Although there is no specific pattern for using wells on the plate, a suggested plate format is shown in Figure 6 below. NOTE: If you do not need to use all the strips at once, 0 place the unused strips back in the plate packet and store at 4°C. Be sure the packet e) e) e) fer Each plate or set of strips should contain is sealed with the desiccant inside. Buf ysat ysat ysat ed L ed L ude L a minimum of two Blk wells, two NSB wells, two B wells, duplicate samples, 0 ein (Cr ein (Clear duplicate buffer control wells for each imidazole concentration, and duplicate ein (Clear Prot Prot T-Prot His- positive controls prepared in imidazole. His- GS Figure 5. His-Protein determination in baculovirus/insect cell lysates expressing 9101112 678 12345 recombinant His-tagged or GST-tagged protein A Blk P10 P2 C5 C13 S8 P10 P2 S8 Blk C5 C13 C8 B NSB S3 S11 C8 NSB S3 S11 P5 P13 P5 P13 Blk - Blank B C3 C11 B P8 NSB - Non-Specific Binding C P8 C3 C11 S6 S14 S6 S14 0 0 B - Maximum Binding 0 S1 P3 P11 C6 C14 S9 P3 P11 S9 D S1 C6 C14 S - Sample C9 S4 S12 S4 S12 P6 P14 C1 C9 P6 P14 E C1 C - Imidazole Buffer Control C4 C12 P - Posi⑱ve Control in C4 C12 P9 S7 P9 S15 S7 S15 P1 P1 F I midazole Buffer P4 P12 P4 P12 C7 C15 S2 C7 C15 S2 G S10 S10 S5 S13 S5 S13 C2 P7 P15 C10 P7 P15 C2 C10 H Figure 6. Sample plate format RESOURCES ANALYSIS 22 23

13 Positive Control Preparation His ELISA TBS Well Negative Sample His-AP Positive Reconstitute the His-Protein ELISA Standard (Item No. 400247) with 400 μl Control Monoclonal Tracer Controls Assay TBS Assay Buffer. The concentration of this solution will be 50 μg/ml. Store this (Imidazole Antibody Buffer Buffer) solution at 4 C; it will be stable for at least two weeks. For longer storage, we ° recommend the bulk standard be aliquoted and stored at -20 ° C or lower. Avoid - - - - - Blk - repeated freeze/thaw cycles. Prepare a separate positive control for every concentration of imidazole. For - NSB l μ 50 - - - l μ 100 example, if samples were eluted in 100 mM, 200 mM, and 300 mM imidazole, prepare a separate positive control in each of these imidazole concentrations. l μ 50 l μ 50 - - - l μ 50 B 0 To prepare the positive control, transfer 10 μl of standard to tubes containing - l Sample μ - - μ l 50 μ l 50 50 1 ml of the varying imidazole buffer concentrations. These tubes are the positive controls. C 50 l 50 μ l μ 50 - - μ - l Addition of the Reagents P - 50 l μ 50 μ - l μ 50 l - 1. TBS Assay Buffer Add 100 μl TBS Assay Buffer to NSB wells. Add 50 μl TBS Assay Buffer to Table 2. Pipetting summary B wells. 0 2. Positive Controls Add 50 μl per well. 3. Negative Control (Imidazole Buffer) Add 50 μl per well. 4. Samples Add 50 μl of sample per well. 5. His-AP Tracer Add 50 μl to each well except the Blk wells. 6. His ELISA Monoclonal Antibody Add 50 μl to each well except the NSB and the Blk wells. RESOURCES RESOURCES 25 24

14 Analysis Incubation of the Plate Cover each plate with plastic film (Item No. 400012) and incubate for 90 minutes NOTE: If the plate reader has not subtracted the absorbance readings of the blank at room temperature on an orbital shaker. wells from the absorbance readings of the rest of the plate, be sure to do that now. Average the absorbance readings of the duplicate (or triplicate) negative 1. Development of the Plate control wells (C) and samples wells (S). 1. Empty the wells and rinse four times with Wash Buffer. Each well should To correct for interference from the imidazole buffer, subtract the Sample 2. be completely filled with Wash Buffer during each wash. Invert the plate , C1 minus i.e. (S) wells from the corresponding negative Control (C) wells ( between wash steps to empty the fluid from the wells. After the last wash, S1, C2 minus S2, etc). This value is the corrected O.D. for each fraction. The gently tap the inverted plate on absorbent paper to remove the residual corrected O.D. of fractions containing His-protein should be greater than Wash Buffer. zero. p NPP Substrate Solution to each well of the plate. Add 200 μl of 2. Positive control (P) wells are not used in the analysis but serve as a diagnostic 3. Cover the plate with plastic film and incubate for approximately 30 minutes tool. The O.D. for the positive control wells MUST be less than the O.D. for at room temperature. The assay typically develops in 30-60 minutes. corresponding imidazole buffer control wells. Reading the Plate Sample Data Wipe the bottom of the plate with a clean tissue to remove fingerprints, dirt, 1. Shown below is an example of data for varying imidazole concentrations etc. containing 0.5 g/ml His-Protein. μ 2. Read the plate at a wavelength between 405 and 420 nm. The absorbance Imidazole Negative Control (C) Sample (S) Wells Corrected O.D. may be checked periodically until the B wells have reached a minimum of 0 Concentration Wells (O.D.) (O.D.) (C minus S) 0.3 A.U. (blank subtracted). The plate should be read when the absorbance (mM) wells in the range of 0.3-1.5 A.U. (blank subtracted). of the B 0 300 0.098 0.064 0.034 200 0.174 0.121 0.053 0.337 0.078 0.259 100 Table 3. Typical results RESOURCES RESOURCES 27 26

15 References Troubleshooting Bornhorst, J.A. and Falke, J.J. Purification of proteins using polyhistidine 1. Recommended Solutions Problem Possible Causes 326 affinity tags. Methods Enzymol. , 245-254 (2000). 2. Variability in the immunodetection et al. Debeljak, N., Feldman, L., Davis, K.L., A. Replace activated A. Trace organic Erratic values; dispersion of of His-tagged recombinant proteins. , 216-223 (2006). 359(2) Anal. Biochem duplicates carbon filter or contaminants in the water source change source of 3. Maxey, K.M., Maddipati, K.R., and Birkmeier, J. Interference in enzyme UltraPure water B. Poor J. Clin. Immunoassay , 116-120 (1992). 15 immunoassays. pipetting/technique High NSB (>10% of B ) A. Re-wash plate and A. Poor washing 0 B. Exposure of NSB wells redevelop to specific antibody Very low B A. Replace activated A. Trace organic 0 carbon filter or contaminants in the water source change source of UltraPure water B. Plate requires additional development time B. Return plate to shaker and re-read C. Dilution error in preparing reagents later Low sensitivity (shift in dose Standard is degraded Replace standard response curve) Analyses of two dilutions of Purify sample prior to Interfering substances are 3 analysis by ELISA a biological sample do not present agree ( i.e. , more than 20% difference) RESOURCES RESOURCES 29 28

16 NOTES 9101112 5678 Warranty and Limitation of Remedy Buyer agrees to purchase the material subject to Cayman’s Terms and Conditions. 34 Complete Terms and Conditions including Warranty and Limitation of Liability information can be found on our website. This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, 12 from Cayman Chemical Company. ©06/27/2017, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. F E D C B A H G Printed in U.S.A. RESOURCES RESOURCES 31 30

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