His Tag ELISA Detection Kit
Contents
1. Evaporation of wells during incubations Perform incubation steps with Plate Sealer in the assay Incorrect incubation times and or temperatures Follow the manual to repeat the assay XI Related Products Toll free Tel 1 877 436 7274 860 Centennial Ave Piscataway NJ 08854 USA Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery e His Tag Antibody pAb Rabbit L00411 e Mouse Anti His mAb MagBeads L00275 e His Tag Antibody Plate L00440 e THE His Tag Antibody mAb Mouse A00186 e THE His Tag Antibody HRP mAb Mouse A00612 e THEIN His Tag Antibody Biotin mAb Mouse A00613 e THEIN His Tag Antibody FITC mAb Mouse A01620 Limited Use Label license This product may be the subject of one or more patents filed by GenScript Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer may not sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for any commercial purposes For commercial use please contact2. pmol ml Duplicate 1 Duplicate 2 Average S 0 0 2 177 2 183 2 180 E 1 5 1 0 088 1 833 1 831 1 832 8 i 3 0 265 1 374 1 418 1 396 8 9 0 796 0 825 0 922 0 874 27 2 389 0 516 0 444 0 480 i an ae a ae a A 81 7 168 0 266 0 289 0 278 His Tagged Protein Concentration ng ml 243 21 504 0 158 0 157 0 158 729 64 513 0 105 0 101 0 103 IX Reagent Compatibility Reagents in test sample may interfere with test result Common detergents and denaturants have been tested for compatibility and interference with the assays Table 3 Reagent compatibility Reagent Recommended Use Triton X 100 lt 1 Imidazole lt 125mM Guanidine HCI lt 30mM Urea lt 0 5 M Deoxycholic Acid lt 1 SDS lt 0 07 EDTA lt 10 mM B ME lt 160mM DTT lt 20 mM Tris pH 7 lt 6 5mM CHAPS lt 5 Tween 20 lt 1 Glycerol lt 1 TBS Fully compatible PBS Fully compatible RIPA Lysis Buffer Fully compatible 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research X Troubleshooting Your Innovation Partner in Drug Discovery Problem Probable Cause Solution Poor Precision Wells are not washed or aspirated properly Make sure the wash apparatus works properly and wells are dry after aspiration Wells have been scratched with pipette tip or washing
3. GenScript at product genscript com GenScript USA Inc 860 Centennial Ave Piscataway NJ 08854 Tel 1 877 436 7274 1 732 885 9188 Fax 732 210 0262 732 885 5878 Email product genscript com Web www genscript com 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery XII Plate Layout Use this plate layout to record standards and samples assayed e a o gt Notes 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com 10 GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery Use this plate layout to record standards and samples assayed e a o gt Notes 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com 11
4. needles Dispense and aspirate solution into and out of wells with caution Particulates are in the samples Remove any particulates by centrifugation prior to the assay Pipette error Check pipette calibration and repeat assay Components are used from other lots or sources Never substitute any components from another kit Components are not brought to room temperature prior to the assay Repeat assay with components that have been equilibrated to room temperature Weak No Signal TMB Substrate are not added or were added at the wrong time Follow the manual to add the TMB Substrate Antibody Tracer is not added or was added at the wrong time Follow the manual to repeat the assay TMB Substrate has been contaminated Use new TMB Substrate Do not add the proper volumes of reagents Repeat the assay with the required volumes in manual Do not incubate the plate for proper time or at proper temperature Do not read the plate immediately after Stop Solution was added Follow the manual to repeat the assay Read the plate within 10 minutes High background Plate is not washed properly Make sure the wash apparatus is functioning properly Make sure wash solution is removed before adding substrate TMB Substrate has been contaminated TMB Substrate is very light sensitive and must be protected from direct light Use new TMB Substrate with same Lot
5. GenScript Transforming Biology Researc h Your Innovation Partner in Drug Discovery His Tag ELISA Detection Kit Technical Manual No TM0635 Version 08292012 Product Name Cat No Size His Tag ELISA Detection Kit L00436 1 plate 8 wells x 12 strips The product is used for rapid and high throughput detection of His tagged proteins The operator should read technical manual carefully before using this product For research use only Not for use in diagnostic procedures 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com Vi VIL VIII Xl XII GenScript Transforming Biology Research Contents RN eil Key eat Enge Seel Ee ege geen Een eae ieee Kit COMUCING ees eege ENEE dea EENEG ENEE Kuele Reagents Equipments Required But Not Supplied c ccccsscccsscessseeeseeees Instruction for EN 1 Sample Dreparation a atni iiyaa 2 Reagent Preparation 3 His tag Plate Br mebatfobt egene eet e e His Tagged Protein Anti His Monoclonal Antibody Incubation Antibody Tracer Incubation v siccisst cccsstisncessseisectaseasecsercssasessesvesncscocarsands Substrate Reaction and Absorbance Measurement sesers Assay Procedure SUMAN y sesers arrire EE EENS Typical Assay KE R age nt Compatibility EH POU SN OU eer aian aine S RE Aa SE aaae Eaa Related PRO CUES sisi fiensssudvnannsssdnubseadsnnnbanysiendsadsines
6. becbeduabuedsisenevbisenozenssaneinhauseanes Plate Layout E 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Your Innovation Partner in Drug Discovery Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery Description His tag is successive histidine H residues and there are mainly three forms HHHHHH 6 x His HHHHH 5 x His HHHH 4 x His Due to its small size less interfere in protein folding weak immunogenicity His tag is the most dominant tag which is widely used in recombinant protein expression The DNA sequence which codes for His tag is usually constructed at N terminus or C terminus of variety of expression plasmids Since His tag has high affinity for NI ions His tagged proteins can be easily purified from bacteria yeast and mammalian cell expression system by Ni resin chromatography Anti His tag antibody is a useful tool for the identification of His tagged proteins with various methods such as western blot immunoprecipitation and flow cytometry GenScript His Tag ELISA Detection Kit a 1 5 hour competition ELISA is developed for rapid and high throughput detection of His tagged proteins There are several potential usages gt Quickly identify the presence of His tagged proteins in samples gt Optimize protein expression by monitoring the His tagged proteins level gt High
7. entration on the horizontal axis The amount of His tagged protein in sample is determined by extrapolating its OD value to the standard curve 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery VII Assay Procedure Summary Add 50 ul of His Tag Standards or samples to each well Add 50 ul of Anti His Monoclonal Antibody to all the wells Wash plates with 260 ul of 1 x Wash Solution four times Add 100 ul of Antibody Tracer Incubate at room temperature for 30 minutes Wash plates with 260 ul of 1 x Wash Solution four times Add 100 ul of TMB Substrate to all the wells Incubate for 10 15 minutes at room temperature Add 50 ul of Stop Solution to all the wells and read the 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com 6 GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery VIII Typical Assay Data The standard curve was provided for demonstration only It should be prepared each time an assay is performed Figure 1 Standard curves of assay results Table 2 Example of assay results Conc of His Tag Standard OD 450 Standard Curve ng ml
8. l 436 60 20 x Wash Solution 40 ml 436 70 TMB Substrate 12 ml 436 40 Stop Solution 6 ml 436 50 His Tag Standard Stock 10 ug ml 500 ul 436 10 Plate Sealer 2 N A User Manual 1 N A IV Storage The unopened kit is stable for at least 12 months if stored at 2 8 C and the opened kit may be stable for up to 1 month at 2 8 C Do not freeze the kit V Reagents Equipments Required But Not Supplied Microtiter plate reader capable of measuring absorbance at 450 nm Automated microplate washer Deionized or distilled water 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery Graduated cylinder to prepare Wash Solution Plastic container to store Wash Solution Tubes to aliquot and dilute samples Precision pipettes to deliver 10 ul 100 pl 200 ul and 1000 ul content 10 ul 100 ul 200 ul and 1000 ul pipette tips Multichannel pipettor Disposable reagent reservoirs Paper towel Laboratory timer Refrigerator to store samples and kit components VI Instruction for Use 1 Sample Preparation When preparing samples for ELISA assay several key factors should be considered e Minimize concentration of certain reagents in the sample For some reagents may interfere with test result read the section of reagents compatibility carefully e Samples should not co
9. ntain any particles precipitates Filter the sample or centrifuge as necessary to remove insoluble materials e For best results the sample should be adjusted to neutral pH 6 8 7 4 2 Reagent Preparation e If any precipitate is found in the 20 x Wash Solution incubate the bottle in water bath up to 50 C with occasional mixing until all the precipitate disappears 1 x Wash Solution Dilute 20 x Wash Solution by 1 19 v v with deionized or distilled water For example dilute 40 ml of 20 x Wash Solution with 760 ml of distilled water to make 800 ml of 1 x Wash Solution The prepared 1 x Wash Solution can be stored at 2 8 C for at least one month 3 His Tag Plate Preparation e Itis recommended that all standards and samples are prepared in duplicate e Make sure the strips are tightly snapped in the plate frame 1 Count the strips for the assay 2 Leave the unused strips in the foil pouch and store at 2 8 C 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery 4 Test Procedure All reagents in the kit and test samples should be equilibrated to room temperature before test Preliminary experiment should be performed to optimize sample dilution Use Assay Diluent for sample dilution The test should not be performed over 25 C His Tagged Protein A
10. nti His Monoclonal Antibody Incubation 1 2 3 4 5 Add 50 ul of His Tag Standards or samples containing His tagged proteins to each well of His Tag Plate Add 50 ul of Anti His Monoclonal Antibody to all the wells Cover the plate with Plate Sealer and incubate at room temperature 20 25 C for 30 minutes Wash the plate with 260 ul of 1 x Wash Solution four times Pat the plate on paper towel to remove residual liquid in the wells Antibody Tracer Incubation 6 7 8 9 Add 100 ul of Antibody Tracer to all the wells Cover the plate with Plate Sealer and incubate at room temperature for 30 minutes Wash the plate with 260 ul of 1 x Wash Solution four times Pat the plate on paper towel to remove residual liquid in the wells Substrate Reaction and Absorbance Measurement 10 11 12 13 14 After adding Stop Solution solution in the wells turns yellow To ensure test stability read the plate at 450 nm immediately after adding Stop Solution If the sample is diluted multiply the interpolated value by the dilution factor to calculate the amount of His tagged proteins in sample Add 100 ul of TMB Substrate to all the wells and incubate at room temperature for 10 15 minutes Add 50 ul of Stop Solution to all the wells to stop the enzyme reaction Read absorbance of the plate on microplate reader at 450 nm Generate a standard curve by plotting the absorbance on the vertical axis versus the His tagged standard conc
11. onding absorbance values are used to construct a standard curve With the standard curve His tagged protein amount present in the unknown sample can be calculated by transforming its absorbance value 860 Centennial Ave Piscataway NJ 08854 USA Toll free Tel 1 877 436 7274 Fax 1 732 210 0262 Email product genscript com Web www genscript com GenScript Transforming Biology Research Your Innovation Partner in Drug Discovery ll Key Features Features Specifications Sensitivity 1 ng ml His tagged proteins Detection Range 1 ng ml 729 ng ml Test Samples N terminal C terminal internal His tagged proteins 4 x His 5 x His 6 x His tagged proteins Mammalian yeast and bacteria cell lysates or cell supernatant Conveniency Provide all reagents required for test Complete test within 1 5 hours Reagent Compatibility Tolerable with various reagents at certain concentration see Table 3 Ill Kit Content The kit provides all reagents and solutions required for His tagged protein detection e His Tag Standard Stock can be used to prepare His Tag Standard with Assay Diluent as an alternative Table 1 Kit components Component Quantity Part No His Tag Plate 1 plate 8 wells x 12 strips 436 80 Anti His Monoclonal Antibody 6 ml 436 20 Antibody Tracer 12 ml 436 30 His Tag Standards 436 2 ml per each standard 0 1 3 9 27 81 243 729 ng ml 11 12 13 14 15 16 17 18 Assay Diluent 60 m
12. throughput screening of stable cell lines expressing His tagged proteins This kit is based on competitive ELISA method His Tag Plate in the kit is a 96 well microtiter plate coated with a His tagged protein with the molecular weight of 12 7 kDa The plate which comprises of 8 wells x 12 strips is demountable When Anti His Monoclonal Antibody and His tagged proteins are added to the well the coated His tagged proteins compete with His Tag Standard His tagged protein with the molecular weight of 11 3 kDa in solution or His tagged protein in sample to interact with Anti His Monoclonal Antibody The higher the concentration of the His tagged protein in solution is the less the antibody bound to the plate will be The Anti His Monoclonal Antibody used in the kit is a mouse anti His tag monoclonal antibody GenScript A00186 Antibody Tracer which is a horseradish peroxidase HRP conjugated Goat anti mouse IgG is used for enzyme reaction His tagged protein Anti His Monoclonal Antibody and antibody tracer form a complex Other unbound molecules can be removed by washing solution The antibody tracer reacts with TMB substrate to develop blue product that turns yellow immediately when the Stop Solution is added which can be measured by microplate reader at 450 nM In optimized test condition each absorbance value is indicated to the individual His tagged proteins amount in solution His tagged protein standards of known concentration and the corresp
Download Pdf Manuals
Related Search