ab Estrogen Receptor Alpha ELISA Kit

Transcription

1 ab Estrogen Receptor Alpha ELISA Kit Instructions for Use For the quantitative measurement of Human, mouse and hamster Estrogen Receptor Alpha concentrations in cell and nuclear extracts. This product is for research use only and is not intended for diagnostic use. Version 3 Last Updated 2 December 2014

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 4 GENERAL INFORMATION 3. PRECAUTIONS 5 4. STORAGE AND STABILITY 5 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 6 7. LIMITATIONS 8 8. TECHNICAL HINTS 8 ASSAY PREPARATION 9. REAGENT PREPARATION CONTROL PREPARATION STANDARD PREPARATION SAMPLE COLLECTION AND STORAGE PLATE PREPARATION 15 ASSAY PROCEDURE 14. ASSAY PROCEDURE 16 DATA ANALYSIS 15. CALCULATIONS TYPICAL DATA ASSAY SENSITIVITY ASSAY SPECIFICITY 19 RESOURCES 19. TROUBLESHOOTING NOTES 22 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Abcam s Estrogen Receptor ELISA kit is an in vitro ELISA (Enzyme- Linked Immunosorbent Assay) designed for accurate quantitative measurement of Human, mouse and hamster Estrogen Receptor Alpha concentrations in cell and tissue nuclear extracts. The Estrogen Receptor Alpha ELISA kit simplifies the measurement of ERα contained in cell and tissue samples by using the Sandwich ELISA method for detecting a protein. This method uses two antibodies that each recognize a distinct epitope on the protein of interest. The kit provides an ELISA plate that is coated with the first antibody, called the Capture Antibody, which is used to capture the protein from the sample. The second antibody, called the Detecting Antibody, is used to detect the protein bound by the Capture Antibody. An HRP-conjugated Secondary Antibody is then used to quantitate the amount of bound Detecting Antibody. Subsequent incubation with developing solution provides an easily quantified colorimetric readout. Estrogen receptor alpha (ERα) belongs to the nuclear receptor (NR) superfamily of structurally related ligand-inducible transcription factors. NRs act in combination with other transcription factors to regulate the expression of gene networks involved in cell growth and development, apoptosis, homeostasis, inflammation, lipid metabolism, the reproductive cycle and other fundamental biological processes. ERα is also a well-established marker of breast cancer hormone sensitivity, and the quantification of estrogen receptors in breast tumors has been routinely performed in clinical laboratories to aid in the selection between hormonal and chemotherapy and also to predict prognosis. Because of ERα s critical role in cell biology, it is important to measure the total amounts of ERα contained in different cell types and tissues. Traditional methods for monitoring ERα protein levels, such as Western blotting, EMSA, immunohistochemistry (IHC) and reporter gene assays, are time consuming and not suitable to high-throughput applications. Discover more at 2

4 INTRODUCTION ER-regulated genes are involved in a variety of cellular pathways that are currently being deciphered by academic and pharmaceutical laboratories for new target discovery. However, there is a lack of standardized assays that measure cellular levels of ER. Once the samples are prepared, this assay is completed in less than 4 hours. As this assay is performed in 96-well plates, a large number of samples can be handled simultaneously, enabling highthroughput automation. This assay is specific for ERα and can be used to detect ERα in as little as 0.6 µg of nuclear extract from MCF-7 cells. The Estrogen Receptor Alpha ELISA Kit has many applications including the study of ER transcriptional activity regulation and protein Structure / function studies of ER and its mutated variants in areas such as osteoporosis, arteriosclerosis and breast cancer. 2. ASSAY SUMMARY Discover more at 3

5 INTRODUCTION Prepare all reagents, samples, and controls as instructed. Plate is supplied pre-coated with capture antibody. Add sample to appropriate wells. Incubate at room temperature. Add primary detection antibody. Incubate at room temperature. Aspirate and wash each well. Add HRP conjugated secondary antibody, which binds the primary antibody. Incubate at room temperature. Aspirate and wash each well. Add developing solution until color develops and then add the stop solution. Immediately begin recording the color development. Discover more at 4

6 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 components at conditions advised immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 9. Reagent Preparation. 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) ERα detecting antibody 1 x 13 μl +2-8 C HRP-conjugated antibody 6 μl (0.2 µg/µl) +2-8 C MCF-7 nuclear extract 40 μl (2.5 µg/µl) -80 C Diluent Buffer 1 x 22 ml -20 C 10X Wash Buffer 1 x 22 ml +2-8 C Developing Solution 1 x 11 ml +2-8 C Stop Solution 1 x 11 ml +2-8 C Pre-Coated 96 Well Microplate (12 x 8 well strips) 96 wells +2-8 C Plate sealer 1 x 1 unit Room temperature Discover more at 5

7 GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Multi-channel pipettor Multi-channel pipettor reservoirs Shaking platform Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference wavelength) These materials are not included in the kit, but will be required for quantification via the production of a standard curve (section 12. Sample Collection and Storage): Recombinant Estrogen Receptor alpha protein These materials are not included in the kit, but will be required for the suggested nuclear extraction protocol (section 11. Standard Preparation): Phosphate Buffered Saline (PBS) 10X PBS For 250 ml, mix: 0.1 M phosphate buffer, ph g Na 2 HPO g KH 2 PO M NaCl 21.9 g 27 mm KCl 0.5 g Adjust to 250 ml with distilled water. Prepare a 1X PBS solution by adding 10 ml 10X PBS to 90 ml distilled water. Sterilize the 1X PBS by filtering through a 0.2 μm filter. The 1X PBS is at ph 7.5. Store the filter-sterilized 1X PBS solution at 4 C. PIB (Phosphatase Inhibitor Buffer) 1X PIB For 10 ml, mix: 25 mm NaF 52 mg 250 mm β-glycerophosphate 0.55 g 250 mm para-nitrophenyl phosphate (PNPP) 1.15 g Discover more at 6

8 GENERAL INFORMATION 25 mm NaVO 3 31 mg Adjust to 10 ml with distilled water. Mix the chemicals by vortexing. Incubate the solution at 50ºC for 5 minutes. Mix again. Store at -20 C. PBS/PIB Prior to use, add 500 μl of PIB into 10 ml of 1X PBS. HB (Hypotonic Buffer) 1X HB For 50 ml, mix: 20 mm Hepes, ph g 5 mm NaF 12 mg 10 μm Na 2 MoO 4 5 μl of a 0.1 M solution 0.1 mm EDTA 10 μl of a 0.5 M solution Adjust ph to 7.5 with 1 N NaOH. Adjust volume to 50 ml with distilled water. Sterilize by filtering through a 0.2 μm filter. Store the filter-sterilized solution at 4 C. Lysis Buffer 1X Lysis Buffer 20 mm Hepes, ph mm NaCl 0.1 mm EDTA 10 mm NaF 10 µm Na 2 MoO 4 1 mm NaVO 3 20% glycerol 10 mm PNPP 10 mm beta-glycerophosphate For 50 ml, mix: 0.24 g 1.17 g 1.5 mg 21 mg 0.12 mg 6.1 mg 10 ml 0.23 g 0.11 g Adjust ph to 7.5 with 1 N NaOH. Adjust volume to 50 ml with distilled water. Store at 4 C. Just before use, make up Complete Lysis Buffer by adding 1 µl of 1 M DTT and 10 µl of Protease Inhibitor Cocktail per ml of Lysis Buffer. Discover more at 7

9 GENERAL INFORMATION 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 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 The Stop Solution is corrosive. Wear personal protective equipment when handling, i.e. safety glasses, gloves and labcoat. Complete removal of all solutions and buffers during wash steps. 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 8

10 ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents and samples to room temperature (18-25 C) prior to use X Wash Buffer Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 10 ml of 1X Wash Buffer required, dilute 1 ml 10X Wash Buffer with 9 ml distilled water. Mix gently to avoid foaming. The 1X Wash Buffer may be stored at 4 C for one week. The Tween 20 contained in the 10X Wash Buffer may form clumps, therefore homogenize the buffer by vortexing for 2 minutes prior to use. 9.2 Antibody Binding Buffer Dilute the ERα detecting antibody to 1:400 and HRPconjugated secondary antibody to 1:1,000 with the Diluent. Use 50 µl of diluted antibody per well. Depending on the particular assay, the signal: noise ratio may be optimized by using higher dilutions of both antibodies. This may decrease the sensitivity of the assay. 9.3 Developing Solution The Developing Solution must be warmed to room temperature for at least 1 hour before use. Prior to use, transfer the amount of Developing Solution required for the assay into a secondary container, avoiding direct exposure to intense light. After use, discard any remaining solution that was transferred into the secondary container. This solution is light sensitive, therefore, we recommend avoiding direct exposure to intense light during storage. The Developing Solution may develop a yellow hue over time. This does not affect product performance. A blue color present in the solution indicates that it has been contaminated and must be discarded. Discover more at 9

11 ASSAY PREPARATION 9.4 Stop Solution Prior to use, transfer the amount of Stop Solution required for the assay into a secondary container. After use, discard remaining Stop Solution. Discover more at 10

12 ASSAY PREPARATION 10. CONTROL PREPARATION Positive control (MCF-7 Nuclear extract) The MCF-7 nuclear extract is provided as a positive control to ensure that the kit reagents are functional. Sufficient extract is provided for 20 reactions. This extract is optimized to give a strong signal when used at 5 µg/well. We recommend aliquoting the extract in 5 µl fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract. Discover more at 11

13 ASSAY PREPARATION 11. STANDARD PREPARATION For optional quantification of the amount of Estrogen Receptor alpha please use the following protocol. The Estrogen receptor alpha protein standard must be reconstituted with the appropriate volume of Diluent Buffer immediately prior to use Reconstitute the Estrogen Receptor alpha standard sample by adding an appropriate volume of Diluent Buffer to produce a 100 ng/μl working stock of recombinant protein. Mix thoroughly and gently Make up a 2.0 ng/µl solution by adding 2.4 µl of the 100 ng/µl working stock to µl of Diluent Buffer. This is the 2,000 pg/ml Standard #1 Solution (see table below). Note: The reconstituted Standard #1 should be discarded after use and not stored for reuse Label six tubes with Standards # Add 60 μl Diluent Buffer into tubes # Prepare Standard #2 by transferring 300 μl from Standard #1 to tube #2. Mix thoroughly and gently Prepare Standard #3 by transferring 300 μl from Standard #2 to tube #3. Mix thoroughly and gently Using the table below as a guide, repeat for tubes #4 through to tube # Standard #8 contains no protein and is the Blank control µl from each tube will be aliquoted to the wells in Step 15.1 of the Assay Procedure (section 15) and will correspond to the following quantities of ERα: 100, 50, 25, 12.5, 6.25, 3.125, and 0.0 ng/well. Discover more at 12

14 ASSAY PREPARATION Standard # Sample to Dilute Volume to Dilute (µl) Volume of Diluent (µl) Starting Conc. (pg/ml) Final Conc. (pg/ml) 1 See Step ,000 2 Standard # ,000 1,000 3 Standard # , Standard # Standard # Standard # Standard # (Blank) Diluent Buffer N/A Discover more at 13

15 ASSAY PREPARATION 12. SAMPLE COLLECTION AND STORAGE Preparation of Nuclear Extract (suggested protocol) For reagent preparation for this protocol see section 6. Materials Required, not Supplied. This procedure can be used for a confluent cell layer of 75 cm 2 (100 mm dish). The yield is approximately 0.5 mg of nuclear proteins for 10 7 cells Wash cells with 10 ml of ice-cold PBS/PIB Add 10 ml of ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer the cells into a pre-chilled 15 ml tube and spin at 300 x g for 5 minutes at 4 C Resuspend the pellet in 1 ml of ice-cold HB buffer by gentle pipetting and transfer the cells into a pre-chilled 1.5 ml tube Allow the cells to swell on ice for 15 minutes Add 50 µl 10% Nonidet P-40 (0.5% final) and mix by gentle pipetting Centrifuge the homogenate for 30 seconds at 4 C in a microcentrifuge Discard the supernatant (which contains the cytoplasm and RNA) carefully without disturbing the pellet. Resuspend the nuclear pellet in 50 µl Complete Lysis Buffer and rock the tube gently on ice for 30 minutes on a shaking platform Centrifuge for 10 minutes at 14,000 x g at 4 C and save the supernatant (nuclear extract) Determine the protein concentration of the extract by using a Bradford-based assay. Storage of the nuclear extract: Aliquot and store at -80 C. Avoid freeze/thaw cycles. Discover more at 14

16 ASSAY PREPARATION 13. 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. For each assay performed, a minimum of 2 wells must be used as blanks, omitting primary antibody from well additions. For statistical reasons, we recommend each sample, control and blank should be assayed with a minimum of two replicates (duplicates). Well effects have not been observed with this assay. If less than 8 wells in a strip are required, cover the unused wells with a portion of the plate sealer while you perform the assay. The content of these wells is stable at room temperature if kept dry and, therefore, can be used later for a separate assay. Store the unused strips in the aluminum pouch at 4 C. Use the strip holder for the assay. Discover more at 15

17 ASSAY PROCEDURE 14. 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. Binding of ERα to the capture antibody 14.1 Sample wells: Add 50 µl of sample diluted in Diluent Buffer to each well to be used. We recommend using 2.5 to 50 µg of nuclear extract diluted in Diluent Buffer per well. A protocol for preparing nuclear extracts is provided below. Control wells: Add 5 µg of the provided MCF-7 nuclear extract diluted in 50 µl of Diluent Buffer to each well to be used (2 µl of extract in 48 µl of Diluent Buffer per well). Blank wells: Add 50 µl Diluent Buffer only per well. OPTIONAL Protein standard wells: Add 50 µl of the appropriate protein standard diluted in Diluent Buffer to each well being used Use the provided adhesive cover to seal the plate. Incubate for 1 hour at room temperature with mild agitation (100 rpm on a rocking platform) Wash each well 3 times with 200 μl 1X Washing Buffer. For each wash, flick the plate over a sink to empty the wells, then tap the inverted plate 3 times on absorbent paper towels. Binding of primary antibody 14.4 Add 50 µl diluted ERα antibody (1:400 dilution in Diluent Buffer) to all wells being used Cover the plate and incubate for 1 hour at room temperature with gentle rocking. Discover more at 16

18 ASSAY PROCEDURE 14.6 Wash the wells 3 times with 200 μl 1X Wash Buffer (as described in Step 14.3). Binding of secondary antibody 14.7 Add 50 µl of diluted HRP-conjugated antibody (1:1000 dilution in Diluent Buffer) to all wells being used Cover the plate and incubate for 1 hour at room temperature with gentle rocking During this incubation, place the Developing Solution at room temperature Wash the wells 4 times with 200 μl 1X Wash Buffer (as described in Step 14.3). Colorimetric reaction Transfer the amount of Developing Solution required for the assay into a secondary container. Add 100 µl Developing Solution to all wells being used Incubate 2-10 minutes at room temperature protected from direct light. Monitor the blue color development in the sample and positive control wells until it turns medium to dark blue. Blank wells should remain faint to light blue. Do not overdevelop Add 100 μl Stop Solution. In presence of the acid, the blue color turns yellow Read absorbance on a spectrophotometer within 5 minutes at 450 nm with a reference wavelength of 655 nm. Blank the plate reader according to the manufacturer s instructions using the blank wells. Discover more at 17

19 DATA ANALYSIS 15. CALCULATIONS If you have generated a standard curve using a recombinant ER protein, average the duplicate readings for each standard, control, and sample and subtract the optical density (OD) obtained from the zero standard. Plot the OD for the standards against the quantity (ng/well) of the standards and draw the best fit curve. The data can be linearized using log/log paper and regression analysis may also be applied. To quantify the amount of ER in the samples, find the absorbance value for the samples on the y-axis and extend a horizontal line to the standard curve. At the intersection point extend a vertical line to the x- axis and read the corresponding standard value. Note: If the samples have been diluted, the value read from the standard curve must be multiplied by the dilution factor. Discover more at 18

20 DATA ANALYSIS 16. TYPICAL DATA This data is provided for demonstration purposes only. Figure 1: Different amounts of nuclear extracts from MCF-7 and MDA-MB-231 cells were analyzed for cellular levels of ERα using the ERα ELISA Kit. 17. ASSAY SENSITIVITY Sensitivity: > 0.6 μg nuclear extract/well. Range of detection: Estrogen Receptor Alpha ELISA kit provides quantitative results from 0.6 to 10 µg of nuclear extract/well. 18. ASSAY SPECIFICITY Cross-reactivity: Estrogen Receptor Alpha ELISA Kit detects ERα from Human, mouse and hamster origin. This assay is not recommended for use with samples from rat origin. Cross-reactivity with other species has not been determined. Discover more at 19

21 RESOURCES 19. TROUBLESHOOTING Problem Cause Solution No signal or weak signal in all wells High background in all wells Uneven color development Omission of key reagent Substrate or conjugate is no longer active Enzyme inhibitor present Plate reader settings not optimal Incorrect assay temperature Inadequate volume of Developing Solution Developing time too short Developing time too long Concentration of antibodies too high Inadequate washing Incomplete washing of wells Well cross-contamination Check that all reagents have been added in the correct order Test conjugate and substrate for activity Sodium azide will inhibit the peroxidise reaction, follow our recommendations to prepare buffers Verify the wavelength and filter settings in the plate reader Bring substrate to room temperature Check to make sure that correct volume is delivered by pipette Increase the development time up to 30 minutes Stop enzymatic reaction as soon as the positive wells turn medium-dark blue Increase antibody dilutions Ensure all wells are filled with Washing Buffer and follow washing recommendations Ensure all wells are filled with Washing Buffer and follow washing recommendations Follow washing recommendations Discover more at 20

22 RESOURCES Problem Cause Solution High background in sample wells No signal or weak signal in sample wells Too much sample per well Concentration of antibodies too high Not enough ERα in the sample added per well ERα is poorly expressed or inactivated in samples Samples are not from correct origin Decrease amount of sample Perform antibody titration to determine optimal working concentration. Start using 1:800 for primary antibody. The sensitivity of the assay will be decreased Increase amount of sample Check ERα expression in the studied sample Refer to cross-reactivity information Discover more at 21

23 RESOURCES 20. NOTES Discover more at 22

24 RESOURCES Discover more at 23

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26 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: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2014 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. RESOURCES 25