ab Androgen Receptor ELISA Kit

Transcription

1 ab Androgen Receptor ELISA Kit Instructions for Use For the quantitative measurement of Human and canine Androgen Receptor concentrations in cell and nuclear extracts. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 26 October 2017

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 SAMPLE COLLECTION AND STORAGE PLATE PREPARATION 13 ASSAY PROCEDURE 13. ASSAY PROCEDURE 14 DATA ANALYSIS 14. TYPICAL DATA ASSAY SENSITIVITY ASSAY SPECIFICITY 16 RESOURCES 17. TROUBLESHOOTING NOTES 19 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Abcam s Androgen Receptor ELISA kit is an in vitro ELISA (Enzyme- Linked Immunosorbent Assay) designed for accurate quantitative measurement of Human and canine Androgen Receptor concentrations in cell and nuclear extracts. The Androgen Receptor ELISA kit uses 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. 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 high-throughput automation. This assay is specific for AR and can be used to detect Androgen Receptor in as little as 0.6 µg of nuclear extract from LNCaP cells. The Androgen Receptor ELISA kit has many applications including the study of AR transcriptional activity regulation and protein structure/function studies of AR in cancer development and progression. Androgen Receptor (AR) 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. AR expression plays an important role in the proliferation of Human Discover more at 2

4 INTRODUCTION prostate cancer and also confers a better prognosis in breast cancer. Because of AR s critical role in cell biology, it is important to measure the total amounts of AR contained in different cell types and tissues. Traditional methods for monitoring AR 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 3

5 INTRODUCTION 2. ASSAY SUMMARY 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) AR detecting antibody 1 x 12 μl +2-8 C HRP-conjugated antibody (0.25 μg/μl) 1 x 12 μl +2-8 C LNCaP nuclear extract (5 μg/μl) 1 x 40 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 RT 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 the suggested nuclear extraction protocol (section 11. Sample Collection and Storage): 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 25 mm NaVO 3 31 mg Discover more at 6

8 GENERAL INFORMATION 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. Ensure complete removal of all solutions and buffers during wash steps. The Stop Solution is corrosive. Wear personal protective equipment when handling, i.e. safety glasses, gloves and labcoat. 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 100 ml of 1X Wash Buffer required, dilute 10 ml 10X Wash Buffer with 90 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 incubating at 50ºC for 2 minutes and mixing prior to use. 9.2 Antibody Binding Buffer Dilute the AR detecting antibody to 1:1,000 and HRPconjugated secondary antibody to 1:1,000 with the Diluent Buffer. 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 Prior to use, place the Developing Solution at room temperature for at least 1 hour. Transfer the amount of Developing Solution required for the assay into a secondary container before aliquoting into the wells. After use, discard remaining Developing Solution. The Developing 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 Developing 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 (LNCaP nuclear extract) The LNCaP nuclear extract is provided as a positive control for RPA activation. Sufficient extract is supplied for 40 reactions per plate. This extract is optimized to give a strong signal when used at 5μg/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. 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 12

14 ASSAY PREPARATION 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. 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 13

15 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, controls, and samples as directed in the previous sections. Binding of AR to the capture antibody 13.1 Sample wells: Add 50 µl of sample diluted in Diluent Buffer to each well to be used. We recommend using 5 to 50 µg of nuclear extract diluted in Diluent Buffer per well. Positive control wells: Add 5 µg of the provided LNCaP nuclear extract diluted in 50 µl of Diluent Buffer to each well to be used (1 µl of extract in 49 µl of Diluent Buffer per well). Blank wells: Add 50 µl Diluent Buffer only per well 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 13.4 Add 50 μl diluted AR antibody (1:1,000 dilution in Diluent Buffer) to all wells being used Cover the plate and incubate for 1 hour at room temperature with mild agitation (100 rpm on an orbital shaker) Wash the wells 3 times with 200 μl 1X Washing Buffer (as described Step 13.3). Binding of secondary antibody Discover more at 14

16 ASSAY PROCEDURE 13.7 Add 50 μl of diluted HRP-conjugated antibody (1:1,000 dilution in Diluent Buffer) to all wells being used Cover the plate and incubate for 1 hour at room temperature with mild agitation (100 rpm on an orbital shaker) During this incubation, place the Developing Solution at room temperature Wash the wells 4 times with 200 μl 1X Washing Buffer (as described Step 13.3). 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 15

17 DATA ANALYSIS 14. TYPICAL DATA This data is provided for demonstration purposes only. Figure 1. Different amounts of nuclear extracts from three human prostate cancer cell lines: LNCap, PC-3 and DU 145 were analyzed for levels of AR protein using the Androgen Receptor ELISA Kit. 15.ASSAY SENSITIVITY Sensitivity: > 0.6 μg nuclear extract/well. Range of detection: Androgen Receptor ELISA kit provides quantitative results from 0.6 to 10 µg of nuclear extract/well. 16. ASSAY SPECIFICITY Cross-reactivity: Androgen Receptor ELISA kit detects AR from Human and canine origin. This assay is not recommended for use with samples from mouse origin. Reactivity with other species has not been determined. Discover more at 16

18 RESOURCES 17. TROUBLESHOOTING Problem Cause Solution No signal or weak signal 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 Check that all reagents have been added in all wells in the correct order Test conjugate and substrate for activity by mixing HRP and Developing Solution together Sodium azide will inhibit the peroxidise reaction, follow our recommendations to prepare buffers Verify the wavelength and filter settings Bring Developing Solution and Stop Solution to room temperature before using Check to make sure that correct volume is delivered by pipette High background in all wells Uneven color development Measurement time too long Concentration of antibodies too high Inadequate washing Incomplete washing of wells Well cross-contamination 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 17

19 RESOURCES Problem Cause Solution High background in sample wells No signal or weak signal in sample wells Too much nuclear extract per well Concentration of antibodies too high Not enough nuclear extract per well Too many freeze/thaw cycles of extract AR is poorly activated or inactivated in nuclear fractions Nuclear extracts are not from correct species Salt concentration too high in binding reaction Decrease amount of nuclear extract down to 1-2 μg/well Perform antibody titration to determine optimal working concentration. Start using 1:1,000 for primary antibody and 1:2,000 for the secondary antibody. The sensitivity of the assay will be decreased Increase amount of nuclear extract. Not to exceed 50 μg/well Aliquot extract into 5 μl aliquots and store at -80 C to avoid multiple freeze/thaws Perform time course for AR activation in the studied cell line Refer to cross-reactivity information on page 5 Reduce amount of extract per well or dialyze extract before use Discover more at 18

20 RESOURCES 18. NOTES Discover more at 19

21 RESOURCES Discover more at 20

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