ab GST tag ELISA Kit

Transcription

1 ab GST tag ELISA Kit Instructions for Use For the quantitative measurement of the GST tag protein expression. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 11 October 2017

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 4 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 5 8. TECHNICAL HINTS 6 ASSAY PREPARATION 9. REAGENT PREPARATION STANDARD PREPARATION SAMPLE PREPARATION PLATE PREPARATION 11 ASSAY PROCEDURE 13. ASSAY PROCEDURE 12 DATA ANALYSIS 14. CALCULATIONS TYPICAL DATA TYPICAL SAMPLE VALUES SPECIES REACTIVITY ASSAY SPECIFICITY 18 RESOURCES 19. TROUBLESHOOTING NOTES 20 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Gluthathione S-Transferase (GST) in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the accurate quantitative measurement of GST tagged protein expression in cell extracts or after purification. The assay employs a GST specific antibody coated onto a well plate strips. Standards and samples are pipetted into the wells and GST present in the sample is bound to the wells by the immobilized antibody. The wells are washed and a primary anti- GST detector antibody is added. After washing away unbound detector antibody an HRP-conjugated secondary detector antibody (HRP Label) specific for the primary detector antibody is pipetted to the wells. The wells are again washed, a HRP substrate solution is added to the wells and color develops in proportion to the amount of GST bound. The developing blue color is measured at 600 nm. Optionally the reaction can be stopped by adding hydrochloric acid which changes the color from blue to yellow and the intensity can be measured at 450 nm. Gluthathione S-transferase (GST) is a 26 kda protein typically used as a fusion partner of recombinant proteins to increase the level of protein expression in bacterial systems, enhance solubility, protect from proteolysis, improve folding and allow purification by affinity chromatography on gluthathione resin. This GST ELISA Kit is an enzyme immunoassay developed for the sensitive detection and quantitation of GST or GST fusion proteins in cells samples or purified tagged proteins. The quantity of GST can be determined in lysates or intermediate purification fractions by comparing their absorbance with that of a known recombinant GST standard curve. The kit has a detection sensitivity limit of 1 ng/ml of GST. Each kit provides sufficient reagents to perform up to 96 assays including standard curve and GST fusion proteins. Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Remove appropriate number of antibody coated well strips. Equilibrate all reagents to room temperature. Prepare all the reagents, samples, and standards as instructed. Add standard or sample to each well used. Incubate at room temperature. Aspirate and wash each well. Add prepared Detector Antibody to each well. Incubate at room temperature. Aspirate and wash each well. Add prepared HRP label. Incubate at room temperature. Aspirate and wash each well. Add HRP Development Solution to each well. Immediately begin recording the color development. Alternatively add a Stop solution at a user-defined time. Discover more at 3

5 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at +2-8ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Reagent and Sample Preparation sections. 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) 20X Buffer 20 ml +2-8ºC Extraction Buffer 15 ml +2-8ºC 10X Blocking Buffer 6 ml +2-8ºC 10X Detector Antibody 700 µl +2-8ºC 10X HRP Label 1 ml +2-8ºC HRP Development Solution 6 ml +2-8ºC GST Standard (2 µg) 1 vial +2-8ºC GST Microplate (12 x 8 antibody coated well strips) 96 wells +2-8ºC Discover more at 4

6 GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Microplate reader capable of measuring absorbance at 600 or 450 nm. Method for determining protein concentration (BCA assay recommended). Deionized water. Multi and single channel pipettes. PBS (1.4 mm KH 2 PO 4, 8 mm Na 2 HPO 4, 140 mm NaCl, 2.7 mm KCl, ph 7.3). Tubes for standard dilution. Stop solution (optional) 1N Hydrochloric acid (HCl). Plate shaker for all incubation steps (optional). Phenylmethylsulfonyl Fluoride (PMSF) (or other protease inhibitors) and/or phosphatase inhibitors. 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Discover more at 5

7 GENERAL INFORMATION 8. TECHNICAL HINTS Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Complete removal of all solutions and buffers during wash steps is necessary to minimize background. As a guide, typical ranges of sample concentration for commonly used sample types are shown below in Sample Preparation (section 11). All samples should be mixed thoroughly and gently. Avoid multiply freeze/thaw of samples. Incubate ELISA plates on a plate shaker during all incubation steps (optional). When generating positive control samples, it is advisable to change pipette tips after each step. This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Discover more at 6

8 ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents to room temperature (18-25 C) prior to use X Wash Buffer Prepare 1X Wash Buffer by adding 20 ml 20X Buffer to 380 ml nanopure water. Mix gently and thoroughly X Incubation Buffer Prepare 1X Incubation Buffer by adding 6 ml 10X Blocking Buffer to 54 ml 1X Wash Buffer. Mix gently and thoroughly. After preparation, the unused 1X Incubation Buffer should be stored at -20 C for up to 6 months 9.3 1X Detector Antibody Prepare 1X Detector Antibody by diluting the 10X Detector Antibody 10-fold with 1X Incubation Buffer immediately prior to use. Prepare 500 µl 1X Detector Antibody for each 8 well strip used X HRP Label Prepare 1X HRP Label by diluting the 10X HRP Label 10- fold with 1X Incubation Buffer immediately prior to use. Prepare 500 µl 1X HRP Label for each 8 well strip used. Discover more at 7

9 ASSAY PREPARATION 10. STANDARD PREPARATION Prepare serially diluted standards immediately prior to use. Always prepare a fresh set of positive controls for every use Reconstitute the GST tag protein standard sample (2 µg) by adding 200 µl nanopure water and vortex vigorously. Allow to sit on ice for 10 minutes. This is the 10 µg/ml Stock Standard. Any remaining stock material should be aliquoted and stored at -80 C Label tubes # Add 150 μl 1X Incubation Buffer into tubes # Prepare 200 ng/ml Standard #1 by adding 980 μl 1X Incubation Buffer into tube #1 and 20 μl of Stock Standard. Mix thoroughly and gently To prepare Standard #2 transfer 150 µl from Standard #1 to tube #2. Mix thoroughly and gently To prepare Standard #3 transfer 150 µl from Standard #2 to tube #3. Mix thoroughly and gently Using the table below as a guide, repeat for tubes #4 through # Standard #8 is a Blank control (0 ng/ml). Add 600 µl 1X Incubation Buffer into tube #8. Discover more at 8

10 ASSAY PREPARATION Standard # Sample to Dilute Volume to Dilute (µl) Volume of Diluent (µl) Starting Conc. (ng/ml) Final Conc. (ng/ml) 1 Stock , Standard # Standard # Standard # Standard # Standard # Standard # (blank) Discover more at 9

11 ASSAY PREPARATION 11. SAMPLE PREPARATION TYPICAL SAMPLE DYNAMIC RANGE - Typical working ranges Sample Type Range GST (pure protein) ng/ml GST fusion protein ng/ml Note: The Extraction Buffer provided with this kit, should only be used with mammalian expression systems. This buffer can be supplemented with phosphatase inhibitors, PMSF and protease inhibitor cocktail prior to use. Supplements should be used according to manufacturer s instructions Preparation of extracts from mammalian cells Collect non adherent cells by centrifugation or scrape to collect adherent cells from the culture flask. Typical centrifugation conditions for cells are 500 x g for 5 minutes at 4ºC Rinse cells twice with PBS Solubilize cell pellet at 2x10 7 /ml in Extraction Buffer Incubate on ice for 20 minutes. Centrifuge at 16,000 x g for 20 minutes at 4 C. Transfer the supernatants into clean tubes and discard the pellets. Assay samples immediately or aliquot and store at -80 C. The sample protein concentration in the extract may be quantified using a protein assay Preparation of extracts from bacterial and yeast cells Disrupt cells by french pressing, sonication, freeze thaw or bead-vortexing according to standard protocols Clarify lysates by centrifuging at 16,000 x g, 4 C for 20 minutes. Discover more at 10

12 ASSAY PREPARATION If fusion protein is insoluble, use either 6 M Guanidine hydrochloride (CH 5 N 3 HCL); 0.1 M NaH 2 PO 4 ; 0.01 M Tris-HCl; ph 8.0 or 7 M Urea (CO(NH 2 ) 2 ); 0.1 M NaH 2 PO 4 ; 0.01 M Tris-HCl; ph Assay samples immediately or aliquot and store at -80 C. The sample protein concentration in the extract may be quantified using a protein assay 12. PLATE PREPARATION The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents. Unused well strips should be returned to the plate packet and stored at +2-8 C. For each assay performed, a minimum of 2 wells must be used as the zero control. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates). Well effects have not been observed with this assay. Discover more at 11

13 ASSAY PROCEDURE 13. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate Prepare all reagents, working standards, and samples as directed in the previous sections Remove unused microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal Add 50 µl of each serially diluted standard (tubes #1-7), blank control (tube #8) or test sample per well The samples should be diluted to within the working range of the assay in 1X Incubation Buffer. As a guide, typical ranges of sample concentration and assay tolerance for commonly used sample buffers are shown below in Data Analysis Cover/seal the plate and incubate for 2 hours at room temperature. If available use a plate shaker for all incubation steps at 300 rpm Aspirate each well and wash, repeat this twice more for a total of three washes. Wash by aspirating or decanting from wells then dispensing 300 µl 1X Wash Buffer into each well as described above. Complete removal of liquid at each step is essential to good performance. After the last wash, remove the remaining buffer by aspiration or decanting. Invert the plate and blot it against clean paper towels to remove excess liquid Add 50 µl 1X Detector antibody to each well used. Cover/seal the plate and incubate for 1 hour at room temperature. If available use a plate shaker for all incubation steps at 300 rpm Repeat the aspirate/wash procedure above. Discover more at 12

14 ASSAY PROCEDURE 13.9 Add 50 µl 1X HRP Label to each well used. Cover/seal the plate and incubate for 1 hour at room temperature. If available use a plate shaker for all incubation steps at 300 rpm Repeat the aspirate/wash procedure above Add 50 µl HRP Development Solution to each empty well and immediately record the blue color development with time in the microplate reader prepared with the following settings: Mode: Wavelength: Time: Interval: Shaking: Kinetic nm up to 15 min 20 sec - 1 min Shake between readings Alternative In place of a kinetic reading, at a user defined, time record the endpoint OD data at (i) 600 nm or (ii) stop the reaction by adding 50 µl stop solution (1N HCl) to each well and record the OD at 450 nm. Analyze the data as described below Analyze the data as described below. Discover more at 13

15 DATA ANALYSIS 14. CALCULATIONS Subtract average zero standard reading from all readings. Average the duplicate readings of the positive control dilutions and plot against their concentrations. Draw the best smooth curve through these points to construct a standard curve. Most plate reader software or graphing software can plot these values and curve fit. A four parameter algorithm (4PL) usually provides the best fit, though other equations can be examined to see which provides the most accurate (e.g. linear, semi-log, log/log, 4 parameter logistic). Read relative protein concentrations for unknown samples from the standard curve plotted. Samples producing signals greater than that of the highest standard should be further diluted and reanalyzed, then multiplying the concentration found by the appropriate dilution factor. Discover more at 14

16 DATA ANALYSIS 15. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. 100 Dynamic Range of the Assay mod/min (600 nm) GST [ng/ml] Figure 1. Example standard curve. GST standard protein was diluted from 400 ng/ml to 6 ng/ml. Background was subtracted from data points prior to log transformation and curve. 16. TYPICAL SAMPLE VALUES SENSITIVITY - Calculated minimum detectable dose = 1 ng/ml (zero dose n= standard deviations). Discover more at 15

17 DATA ANALYSIS RECOVERY (Sample spiking in representative sample matrices) Sample Type Average % Recovery Range (%) 50% mammalian extraction buffer % Guanidine Buffer % Urea buffer % Gluthathione buffer % Yeast extraction buffers If measuring GST tagged on proteins purified by gluthathione resins, dilute standard curve in 0.1% glutathione buffer and dilute eluate by 1:1,000 factor. Gluthathione buffer can decrease significantly the sensitivity of the assay. It is recommended to dialyze the protein prior to assay measurement. LINEARITY OF DILUTION P53-GST recombinant protein (ng/ml) ab43615 % Expected Value PRECISION Intra- Assay Inter- Assay n= 5 3 %CV Discover more at 16

18 DATA ANALYSIS ASSAY TOLERANCE Tolerance of this assay was tested with buffers typically used in recombinant protein chemistry sample preparation: Buffer Name Composition Maximum concentration tolerated by assay Effect at higher concentrations Guanidine buffer Urea buffer Gluthathione buffer Mammalian detergent Yeast detergents 6 M GuHCl 0.1 M NaH2PO M Tris-HCl ph M urea 0.1 M NaH2PO M Tris-HCl ph mM Tris-HCL 50mM reduced Glutathione ph 8.0 Provided the kit Commercially available with 5% Inhibition 5% Inhibition 0.1% Inhibition 50% Inhibition 0.1% Agonist 17. SPECIES REACTIVITY This kit detects GST protein in mammalian samples only. Other species have not been tested. Discover more at 17

19 DATA ANALYSIS 18. ASSAY SPECIFICITY This kit is reactive with Schistosoma japonicum GST fused to His-tag at N-terminus (ab89494) and it was tested with recombinant full length protein (Human) p53 with N terminal GST tag (ab43615). Note: it is possible that improper folding of GST or the presence of a fusion partner at the C-terminus may prevent GST binding in this kit. Both capture and detector antibodies were also evaluated with the ab89494 and ab43615 by western blot. L2 250kDa 150kDa 100kDa 75kDa 50kDa 37kDa 25kDa 20kDa 10kDa Figure 2. Validation by Western blot. Capture (left) and detector (right) antibodies were tested by western blot against GST recombinant protein ab89494 (lane 1) and p53 tagged to GST protein ab43615 (lane 2). Discover more at 18

20 RESOURCES 19. TROUBLESHOOTING Problem Cause Solution Inaccurate Pipetting Check pipets Poor standard curve Improper standard dilution Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing Low Signal Large CV Low sensitivity Incubation times too brief Inadequate reagent volumes or improper dilution Plate is insufficiently washed Contaminated wash buffer Improper storage of the ELISA kit Ensure sufficient incubation times; change to overnight standard/sample incubation Check pipettes and ensure correct preparation Review manual for proper wash technique. If using a plate washer, check all ports for obstructions Make fresh wash buffer Store the reconstituted protein at -80 C, all other assay components +2-8 C. Keep substrate solution protected from light. Discover more at 19

21 RESOURCES 20. NOTES Discover more at 20

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24 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中中中中 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2017 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. RESOURCES 23