ab For the qualitative measurement of phosphorylated Ser2448 of mtor protein in cell and tissue lysates.

Transcription

1 ab mtor (pser2448) ELISA Kit Instructions for Use For the qualitative measurement of phosphorylated Ser2448 of mtor protein in cell and tissue lysates. This product is for research use only and is not intended for diagnostic use. Version Last Updated 8 June 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 POSITIVE CONTROL 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 17 RESOURCES 19. TROUBLESHOOTING NOTES 21 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Abcam s mtor pser2448 in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the qualitative measurement of Ser2448 of mtor protein in cell and tissue lysates. Mammalian target of rapamycin (mtor) is a serine/threonine protein kinase part of two distinct signaling complexes, mtorc1 and mtorc2. These two complexes share four proteins (mtor, mlst8, DEPTOR, Tti1/tel2), with only mtorc1 containing Raptor and PRAS40 and mtorc2 containing Rictor, msin1 and Protor1/2. The complex mtorc1 (rapamycin sensitive complex) coordinates inputs from growth factors, stress, energy status, oxygen and amino acids levels to control processes such as protein and lipid synthesis and autophagy. The complex mtorc2 is insensitive to nutrients and rapamycin, but it responds to insulin signaling. It also controls ion transport and cell shape by targeting serum/glucocorticoid protein kinase (SGK1) and protein kinase (PKC-α) respectively. The canonical regulation of mtorc1 occurs through the TSC/Rheb pathway which receives signals from AKT, AMPK and IKKβ to activate the complex. Phosphorylation of mtor at Ser2448 is carried out directly by AKT kinase as well as p70s6 kinase acting as a feedback signal. Phosphorylation at this site is a biomarker for the activation state of the PI-3 kinase pathway as well as the activation status of mtor. Activation of mtor leads to phosphorylation of PRAS40, raptor and DEPTOR and the consequential activation of mtorc1. Deregulated signaling of mtor has been implicated in diseases such as cancer, metabolic syndrome, neurodegeneration and aging. Constitutive activation of PI3K-mTORC1 signaling in cancer cells inhibits autophagy, deregulates protein synthesis via 4E-BP1/eIF4E and increases de novo lipid synthesis via SREBP1. Similarly mtor signaling is a key factor in the regulation of tissue metabolism in the normal and nutrient overload state affecting the hypothalamus, adipose tissue, the liver, skeletal muscle and pancreas. 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 positive control 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 and incubate for 30 minutes. Add Stop solution and record end point. 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 4ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in sections 9 & MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) 10X Wash Buffer 40 ml 4ºC Extraction Buffer 15 ml 4ºC 10X Blocking Buffer 6 ml 4ºC mtor Microplate 96 wells 4ºC 10X mtor (pser2448) Detector Antibody 700 µl 4ºC 10X HRP Label 1 ml 4ºC HRP Development Solution 12 ml 4ºC Stop Solution 12 ml 4ºC 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 KH2PO4, 8 mm Na2HPO4, 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 (sodium fluoride or a cocktail containing sodium orthovanadate, sodium molybdate, sodium tartrate and imidazole). 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. 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 40 ml 10X Wash Buffer to 360 ml nanopure water. Mix gently and thoroughly. 9.2 Supplemented Extraction Buffer Supplement appropriate volume of extraction buffer with protease and phosphatase inhibitors immediately prior to use X Supplemented Incubation Buffer Prepare Incubation Buffer by adding 4 ml 10X Blocking Buffer to 36 ml 1X Wash Buffer. Supplement this buffer with a phosphatase inhibitor cocktail and/or 10 mm Sodium fluoride. Mix gently and thoroughly X Incubation Buffer Prepare Incubation Buffer by adding 2 ml 10X Blocking Buffer to 18 ml 1X Wash Buffer. Mix gently and thoroughly X mtor pser2448 Detector Antibody Prepare 1X mtor pser2448 Detector Antibody by diluting the 10X mtor pser2448 Detector Antibody 10-fold with 1X Supplemented Incubation Buffer immediately prior to use. Prepare 500 µl 1X mtor pser2448 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. After opening, the unused 10X Blocking buffer and Incubation Buffer should be stored at -20 C. Discover more at 7

9 ASSAY PREPARATION 10. POSITIVE CONTROL PREPARATION Prepare serially diluted positive controls immediately prior to use. Always prepare a fresh set of positive controls for every use. The levels of phosphorylated Ser2448 of mtor are low in many standard cell lines and require induction with pharmacological treatment (see Specificity section below). In cell lines where levels of are low, mtor Ser2448 may be phosphorylated with calyculin A treatment (e.g. HeLa cells treated with 50 nm calyculin for 15 minutes or NIH3T3 cells treated with 50 ng/ml of rpdgf for 30 minutes in serum free media). Use a positive control sample to prepare the dilution series. The relative levels of pser2448 of mtor in other experimental samples can be interpolated from within this positive control sample series Prepare a positive control sample lysate or extract as directed in sections 11. Dilute the positive control sample to an upper concentration limit of the working range of the assay in the 1X Supplemented Incubation Buffer used to dilute other experimental samples = Standard # Label tubes #2-7: Add 150 μl 1X Supplemented Incubation Buffer into each tube Add 150 μl Standard #1 to tube #2 and mix thoroughly = Standard # Transfer 150 μl from Standard #2 to tube #3, mix thoroughly = Standard # Repeat for Tubes #4 through # Use the diluent as the zero standard tube labeled #8. Discover more at 8

10 ASSAY PREPARATION 11. SAMPLE PREPARATION TYPICAL SAMPLE DYNAMIC RANGE - Typical working ranges Sample Type Range HeLa cells treated with calyculin A MCF7 cells treated with Phorbol 12-myristate 13-acetate (PMA) µg/ml µg/ml NIH3T3 cells treated with PDGF µg/ml 11.1 Preparation of extracts from cell pellets 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 Supplemented Extraction Buffer Incubate on ice for 20 minutes. Centrifuge at 18,000 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 (avoid freeze/thaw cycles). The sample protein concentration in the extract may be quantified using a protein assay Preparation of extracts from adherent cells by direct lysis (alternative protocol) Remove growth media and rinse adherent cells 2 times in PBS Solubilize the cells by addition of Supplemented Extraction Buffer directly to the plate (use 750 µl ml Extraction Buffer per confluent 15 cm diameter plate). Discover more at 9

11 ASSAY PREPARATION Scrape the cells into a test tube and incubate the lysate on ice for 15 minutes. Centrifuge at 18,000 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 (avoid freeze/thaw cycles). The sample protein concentration in the extract may be quantified using a protein assay Preparation of extracts from tissue homogenates Tissue lysates are typically prepared by homogenization of tissue that is first minced and thoroughly rinsed in PBS to remove blood (dounce homogenizer recommended) Homogenize 100 to 200 mg of wet tissue in 500 µl 1 ml of the Supplemented Extraction Buffer. For lower amounts of tissue adjust volumes accordingly Incubate on ice for 20 minutes. Centrifuge at 18,000 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 (avoid freeze/thaw cycles). The sample protein concentration in the extract may be quantified using a protein assay Discover more at 10

12 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. Unused plate strips should be returned to the plate packet and stored at 4 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. Contents of each well can be recorded on the template sheet included in the Resources section. 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 and samples as directed in the previous sections The samples should be diluted within the working range of the assay in 1X Supplemented Incubation Buffer. As a guide, typical ranges of sample concentration are shown above (section 11). Add 50 µl of each sample. Also include a 1X Incubation buffer as a background control Cover/seal the plate and incubate shaking at 400rpm for 2 hours at room temperature Wash each well three times Add 50 µl of 1X mtor pser2448 Primary Detector Antibody (step 9.5) to each well used. Cover/seal the plate and incubate shaking at 400 rpm for 1 hour at room temperature Wash each well 3 times Add 50 µl 1X HRP Labeled Secondary Detector antibody in 1X Incubation Buffer (step 9.6) to each well used. Cover/seal the plate and incubate at 400 rpm for 1 hour at room temperature Wash each well three times Add 100 µl HRP Development Solution to each empty well and incubate shaking at 200 rpm for 30 minutes at room temperature away from the light Add 100 µl of Stop solution to each well and read end point at 450 nm in the microplate reader. Discover more at 12

14 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, asymmetric sigmoidal 5 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 13

15 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. Standard Curve Measurements Conc. O.D. 450 nm (µg/ml) Mean O.D. 450 nm Figure 1. Dynamic Range of the assay. The extract was prepared with HeLa cells treated with 50 nm of calyculin A for 3 hours as described in section Data was plotted on an XY graph in the log scale and an asymmetric sigmoidal 5 parameter logistic algorithm formula was used for curve fitting. Discover more at 14

16 DATA ANALYSIS 16. TYPICAL SAMPLE VALUES SENSITIVITY - Calculated minimum detectable dose = 8 µg/ml (zero dose n= standard deviations) using HeLa cells treated with 50 nm of calyculin A for 15 minutes. RECOVERY (Sample spiking in representative sample matrices) Sample Type Average % Range % Recovery Recovery 50% Culture Media (8 Dilutions) % Serum (8 Dilutions) % Extraction Buffer (8 Dilutions) LINEARITY OF DILUTION Linearity of dilution was determined by comparing a dilution series of extracts prepared from HeLa cells treated with 50 nm calyculin A for 3 hours to extracts prepared with MCF7 cells treated with 200 µm PMA for 1 hour (starting protein concentration at 500 µg/ml) MCF7 PMA Loading Dilution factor (µg/ml) % Expected Value 1: % 1: % 1: % 1: % 1: % PRECISION Intra-Assay Inter-Assay n= 24 3 %CV 4.3% 1.8% Discover more at 15

17 DATA ANALYSIS 17. SPECIES REACTIVITY This kit detects mtor pser2448 in human and mouse samples. Cross reactivity to mouse samples was determined with the mouse cell line NIH3T3 treated with 50 ng/ml of PDGF A/B recombinant protein for 30 minutes after overnight serum starvation. Figure 4 shows performance of this kit on NIH3T3 PDGF treated against DMSO control. Figure 2. The mtor pser2448 ELISA cross reacts with mouse samples. NIH3T3 cells were treated with 50 ng/ml of PDGF recombinant protein after serum starvation or BSA control for 30 minutes at 37 C. Samples were prepared according to section A protein titration of PDGF treated cells was loaded in triplicate (left panel) alongside 4 replicates of both BSA and PDGF treated cells at 500 µg/ml. BSA and PDGF samples were interpolated from the standard curve and results are shown on the right panel. PDGF increases the levels of phosphorylation at Ser2448 by 2 fold. Discover more at 16

18 DATA ANALYSIS 18. ASSAY SPECIFICITY The specificity of the assay to measure mtor phosphorylated at Ser2448 was demonstrated with the use of λ protein phosphatase (λ Ppase) on calyculin A treated HeLa cells. The relative levels of the phosphorylated Ser2448 in extracts decreased dramatically when treated with 1/100 dilution of the enzyme (Figure 3). This result matched well with a parallel Western blot analysis (using the kit s detector antibody) of the same λ protein phosphatase treated sample used on the ELISA format (Figure 4). HeLa cells treated with 50 nm of calyculin for 15 minutes were lysed with the kit s extraction buffer without phosphatase inhibitor supplements and lysate was divided into two vials: Control and λ Ppase. The control vial was supplemented with 10mM sodium fluoride and a cocktail containing sodium orthovanadate, sodium molybdate, sodium tartrate and imidazole (not provided with the kit) and left on ice. The λ Ppase aliquot was diluted 1:4 in 50 mm Hepes, 100 mm NaCl, 2 mm DTT, 0.01 % Brij 35 ph 7.5 and 1mM MnCl 2 (not provided with the kit). Lambda phosphatase was added at 1/100 dilution and the vial was incubated at 34 C for 45 minutes. At the end of the treatment, all samples were divided into two further vials, one was diluted in SDS loading buffer and analyzed by Western blotting whereas the other was diluted in incubation buffer and analyzed with the kit. Discover more at 17

19 DATA ANALYSIS Figure 3. The mtor pser2448 ELISA specifically measures the phosphorylated protein. Calyculin treated HeLa extracts were left untreated (control) or treated with 1:100 dilution of λ Ppase at 34 C. Samples were loaded at 500, 125 and 30 µg/ml on the plate and measured following the kit s protocol. Treatment of calyculin treated HeLa extracts with λ Ppase decreases the signal by 12 fold at the highest loading. Discover more at 18

20 DATA ANALYSIS Figure 4. Western blot using capture anti-mtor and detector anti-mtor pser2448. The detector antibody used in this kit specifically detects the phosphorylated mtor as determined by Western blotting. Samples were loaded on an 8% polyacrylamide gel as follows: (1) Marker (2) calyculin treated HeLa lysate, (3) calyculin treated HeLa lysate dephosphorylated with 1:100 dilution of λ Ppase at 34 C, (4) Hek293T cell lysate. Samples were then diluted in SDS-PAGE buffer and loaded at 40 µg/well. Membranes were blocked with 20% Blocking Buffer ab in TBST for 1 hour and incubated with either the detector antibody mtor pser2448 or the capture antibody against total mtor in 10% blocking buffer ab in TBST overnight. Labeling was carried out with secondary antibodies conjugated to HRP. Treatment with calyculin A in HeLa cells induces phosphorylation at Ser2448 and λ Ppase completely dephosphorylates mtor. Discover more at 19

21 RESOURCES 19. TROUBLESHOOTING Problem Cause Solution Low Signal Large CV Incubation times too brief Inadequate reagent volumes or improper dilution Active phosphatases in the extract Incubation times with HRP Development Solution too brief Plate is insufficiently washed Ensure sufficient incubation times; change to overnight standard/sample incubation Check pipettes and ensure correct preparation Add protein phosphatase inhibitors into the Extraction Buffer and into the Incubation Buffer used for the sample and the detector antibody dilutions Ensure sufficient incubation time till blue color develops prior addition of Stop solution Review manual for proper wash technique. If using a plate washer, check all ports for obstructions Low sensitivity Contaminated wash buffer Improper storage of the ELISA kit Prepare fresh wash buffer Store all assay components 4 C. Keep substrate solution protected from light Discover more at 20

22 RESOURCES 20. NOTES Discover more at 21

23 RESOURCES Discover more at 22

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: ( 中國聯通 ) 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